Activated GTPase-based assays and kits for the diagnosis of sepsis and other infections

ABSTRACT

In one embodiment, the invention provides a method of diagnosing sepsis or a virus-related infection (often a viral hemorrhagic fever infection) in a subject by detecting and measuring the level of a set of sepsis and virus infection-associated-GTPase biomarkers in a sample obtained from the subject using multiplexed flow cytometry. Related kits are also provided. In a preferred embodiment, the invention provides point of care diagnostic methods for determining an early stage sepsis or the severity of a virus infection, especially in a hospital or other setting.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/626,536 filed on Feb. 19, 2015, now U.S. Pat. No.10,261,084, which claims priority from U.S. Provisional PatentApplication Ser. No. 61/941,604, entitled “Rapid, Effector-Based,Flow-Cytometry Assay for Activated GTPases”, and filed 19 Feb. 2014. Thecomplete contents of these two priority patent applications are herebyincorporated by reference in their entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant No.1R21NS066435 awarded by the National Institute of Neurological Disordersand Stroke (NINDS); Grant Nos. R21NS066435, R03A1082130, R03A1092130,R21NS066429 and IP50GM085273 awarded by the National Institute of Health(NIH); and Grant No. MCB0956027 awarded by the National ScienceFoundation (NSF). Consequently, the government has certain rights in theinvention.

FIELD OF THE INVENTION

In one embodiment, the invention provides a method of diagnosing abacterial infection (sepsis) in a subject by using multiplexed flowcytometry to detect and measure the level of active GTPase enrichmentcaused by bacterial infection. Related kits are also provided.

In a preferred embodiment, the invention provides point of careactivated GTPase-based diagnostic methods and related kits fordetermining an early stage sepsis, especially in a hospital setting.

More specifically, certain embodiments provide a rapid assay formeasuring the cellular activity of small GTPases in response to aspecific stimulus. Effector functionalized beads are used to quantify inparallel multiple, GTPbound GTPases in the same cell lysate by flowcytometry. In particular biologically relevant examples, different Ras(HRas, Rap1), Rho (Rac1, Cdc41, RhoA) and Rab (Rab 5) family GTPases areshown for the first time to be involved in a concerted signaling cascadedownstream of receptor ligation by Sin Nombre hantavirus. In anothersetting, the preclinical onset of sepsis was manifested by theenrichment of active GTPases (Rho, Rap1 or Rac1) measured in serialplasma samples taken from trauma patients who were clinically diagnosedwith bacterial infection.

BACKGROUND OF THE INVENTION

The Ras superfamily of small GTPases is comprised of five major groups:Ras, Rho, Rab, Arf and Ran that regulate many aspects of cell behavior.The Ras (e.g. H-Ras, K-Ras, R-Ras and Rap 1) and Rho subfamily (e.g.Cdc42, Rac1 and RhoA) of GTPases synergistically regulate signalingpathways that originate from extracellular stimuli, to yield overlappingsets of cellular phenotypes, such as proliferation, differentiation, andremodeling of the cytoskeleton. The GTPases function by cycling betweenactive GTP-bound and inactive GDP-bound states. Guaninenucleotide-exchange factors (GEFs), GTPase-activating proteins (GAPs)and guanine nucleotide-dissociation inhibitors (GDIs) (Guilluy et al.,2011; Jaffe and Hall, 2005) control the activity of the GTPases. GEFsactivate Rho proteins by catalyzing the exchange of GDP for GTP, whileGAPs inactivate the proteins by stimulating intrinsic GTPase activity.GDI inhibits the activation of Rho GTPases by sequestering them in thecytosol away from membranes. Activated GTPases interact with specificdownstream-effector proteins to yield definite physiological responsesin response to the upstream stimuli. Ras and Rho family GTPases functionas components of a broader signaling network and are interconnectedacross overlapping signaling pathways that involve positive and negativefeedback loops.

The superfamily of GTPases has numerous cellular effects that aredysregulated in disease. Ras (35 members) primarily involved insignaling and cancer. Rho (23 members) GTPases are primarily involved incell motility, infection and cancer among others. Rab (70 members)GTPases are primarily involved in intracellular transport, cancer,infectious disease, genetic disease and downstream growth factorsignaling Ran (1 member) nuclear import, cellular differentiation, Arf(30 members) intracellular transport, infectious disease, humanciliopathies and retinopathies. The dysregulation of these systems canbe measured as an increase in the enrichment of active GTPases caused byfactors in patient samples. Active GTPases preferentially bind tospecific cognate effector molecules that are immobilized on beads, thusproviding evidence as to the dysregulation of the systems involved.

The interactions of viruses and host cells is known to elicit theactivation of multiple GTPases to promote the cytoskeletal remodelingrequired for breaching inter- and intracellular cellular barriers toinfection as well as intracellular trafficking of internalized virionsto allow replication. Most studies investigating the role of GTPases inviral interactions with host cells use traditional methods of activeGTPase pull-down and detection by Western blot, which are slow, laborintensive and require large amounts of starting material. Newer,commercially available plate-based effector binding assays for detectingactivated GTPases known as GLISA (Cytoskeleton, Inc.) require lessmaterial than western blot based assays, yet are still labor intensive;requiring freezing of aliquots, protein assays to ensure linearity andnumerous binding and washing steps. Accordingly, most studies tend tofocus on a limited subset of GTPases, which presents significantlimitations when one wants to examine the broader spectrum of cellsignaling space impinged upon by viral activity. Based on theseconsiderations, we have developed a rapid, and quantitative flowcytometry-compatible, bead-based effector binding assay to analyze, inparallel, multiple GTPases that are activated in a single virus-infectedcell sample.

Sepsis is a disease that now affects more than 900,000 patients with anestimated mortality rate of 30% in the US.^(22-25A) Annual costs areestimated to exceed $20 billion.^(26A) Severe trauma patients whosurvive the initial injury are at risk of developing sepsis syndrome andmultiple organ failure.^(24A, 25A, 27A) Systemic microvascular leakage,most likely due to the release of inflammatory, coagulation andfibrinolysis factors, is a signature of sepsis in trauma patients.^(22A)An improved understanding of the clinical mechanisms of sepsis,including the roles of pathogens, sites of injury and patientheterogeneity, is urgently needed to enable better prevention,diagnosis, and treatment.^(22A) The goal of this project is to addressthe need for timely and accurate differential diagnosis of sepsis andSIRS due to sterile inflammation. The pathophysiology of sepsis involvesnearly all cell types, tissues, and organ systems, and has so far beenassociated with about 180 distinct potential biological markers.^(4-8A)These markers are organized as follows: vasoactive amines, vasoactivepeptides, fragments of complement components, lipid mediators,cytokines, chemokines, and proteolytic enzymes involved in thecoagulation and fibrinolytic system.^(9A) This level of heterogeneitycontinues to confound efforts to discover universally applicable models.

A heterogeneous patient population and a diverse ensemble of pathogenicbacteria highlight a cardinal problem in defining the pathogenesis ofsepsis.^(1-3A) So far, about 180 potential biological markers of sepsisare known.^(4-8A) Their broad-spectrum applicability to sepsis and otherpathologies has limited their early diagnostic utility.^(9A) Newapproaches accounting for the complexity of the inflammatory responseand changes that occur during the course of sepsis are needed.

Small GTPases and their regulators, as they actuate and fine-tunepivotal molecular pathways, constitute vulnerable nodes of the cell.Their activities are associated with a a diverse range of biologicalfunctionality in health and disease, such as cancer[1-3], cardiovasculardiseases or developmental diseases [6], and infections [4,5] In disease,GTPAse signalng pathways are diverted during the onset or progression ofthe disease, and disorders in which the expression, regulation, orfunction of regulators is directly impaired by mutations. These includecongenital diseases in which GTPase regulators carry missense mutationsthat impair their biochemical properties, and infections in whichpathogens have created new regulators of their own to take command ofhost pathways. For some of these diseases, understanding the biochemicalbasis may help in discovering pharmaceuticals to correct these defects.Through evolution, bacterial pathogens ha % e evolved a batter of toxinsand virulence factors that target small GTPases that attenuate GTPasesfunctions, in order to facilitate host entry and dissemination.

-   1. Vega. F. M.; Ridley, A. J. Rho gtpases in cancer cell biology.    FEBS letters 2008, 582, 2093-2101.-   2. Iden, S.; Collard, J. G. Crosstalk between small gtpases and    polarity proteins in cell polarization. Nature reviews. Molecular    cell biology 2008, 9, 846-859.-   3. Agola, J.; Jim, P.; Ward, H, Basuray, S.; Wandinger-Ness, A. Rab    gtpases as regulators of endocytosis, targets of disease and    therapeutic opportunities. Clinical genetics 2012.-   4. Lemichez, E.; Aktories, K. Hijacking of rho gtpases during    bacterial infection. Experimental cell research 2013, 319,    2329-2336.-   5. Aktories, K.; Schmidt, G. A new turn in rho gtpase activation by    Escherichia coli cytotoxic necrotizing factors. Trends in    microbiology 2003, 11, 152-155.-   6. Cherfils, J.; Zeghouf, M. Regulation of small gtpases by gefs,    gaps, and gdis. Physiological reviews 2013, 93, 269-309.

SUMMARY OF THE INVENTION

Early diagnosis of bacterial infection is critical for effectiveintervention in sepsis. We have employed active real time surveillanceof factors that are enriched in the blood of trauma patients inproteomic and computational network analyses that link temporal changesin protein expression and hemostasis to identify novel clusters ofpotential targets for effective treatments, based on early detection andthe development of new molecular-based therapies.

More specifically, we have determined the preclinical onset of infectionby sensing bacteria-initiated hemostatic impairment^(3A, 10-13-A) andactivation of RhoGTPases.^(14-20A) Hemostasis is a critical process thatacts to seal breaches in the vascular system (clotting) either toprevent bleeding, and/or to block access for pathogens to the vascularsystem. Some bacteria are able to promote their dissemination byinitiating fibrinolysis.^(3A, 10-13A, 221A) Thrombin is a versatileserine protease that is generated at sites of vascular injury andconverts fibrinogen into fibrin monomers. [1-7] Endogenous fibrinolysisis a protective mechanism against lasting arterial and venous thromboticocclusion, caused by fibrin clots. [8] Many gram-positive andgram-negative bacterial species can shift the fibrinolytic balance byinducing an increase in the concentration of plasmin in septic patients.[9-12] Fibrinolysis allows bacteria to disseminate beyond fibrin clotbarriers that are meant to limit bacterial spread. [11-15] In addition,bacteria use virulence factors to modulate GTPase activity of hostcells.[16-22] Therefore, our goal is to identify the onset of sepsis byanalyzing, in parallel, the impairment of hemostasis and modulation ofsmall molecule GTPases in serial blood samples collected from traumapatients with clinically confirmed sepsis. Our approach is to combineinterdisciplinary expertise and tools to meet the goal of enablingbetter prevention, diagnosis, and treatment of sepsis. We have developedthe necessary tools for measuring hemostatic impairment and activity ofmultiple RhoGTPases. [23-25] The Gtrap multiplex system developed is aunique tool that enables rapid measurement of the activity of GTPbinding Rho proteins.[24].

-   1. Levi, M.; Keller, T. T.; van Gorp, E.; ten Cate, H. Infection and    inflammation and the coagulation system. Cardiovascular research    2003, 60, 26-39.-   2. Kumar, P.; Shen, Q.; Pivetti, C. D.; Lee, E. S.; Wu, M. H.;    Yuan. S. Y. Molecular mechanisms of endothelial hyperpermeability:    Implications in inflammation. Expert Rev Mol Med 2009, 11.-   3. Escolar, G.; Bozzo. J.; Maragall, S. Argatroban: A direct    thrombin inhibitor with reliable and predictable anticoagulant    actions. Drugs Today 2006, 42, 223-236.-   4. Carbajal, J. M.; Gratrix, M. L.; Yu, C. H.; Schaeffer, R. C., Jr.    Rock mediates thrombin's endothelial barrier dysfunction. American    journal of physiology. Cell physiology 2000, 279. C195-204.-   5. Anwar, K. N.; Fazal, F.; Malik, A. B.; Rahman, A.    Rhoa/rho-associated kinase pathway selectively regulates    thrombin-induced intercellular adhesion molecule-1 expression in    endothelial cells via activation of i kappa b kinase beta and    phosphorylation of rela/p65. J Immunol 2004, 173, 6965-6972.-   6. Sosothikul. D.; Seksarn, P.; Pongsewalak, S. Thisyakorn, U.;    Lusher, J. Activation of endothelial cells, coagulation and    fibrinolysis in children with dengue virus infection. Thrombosis and    haemostasis 2007, 97.627-634.-   7. Laine, O.; Makela, S.; Mustonen, J.; Huhtala, H.; Szanto, T.;    Vaheri, A. Lassila, R.; Joutsi-Korhonen, L. Enhanced thrombin    formation and fibrinolysis during acute puumala hantavirus    infection. Thrombosis research 2010, 126, 154-158.-   8. Dubis, J.; Witkiewicz, W. The role of thrombin-activatable    fibrinolvsis inhibitor in the pathophysiology of hemostasis. Adv Cin    Exp Med 2010, 19, 379-387.-   9. McAdow, M.; Kim, H. K.; DeDent, A. C.; Hendrickx, A. P. A.;    Schneewind, O. Missiakas, D. M. Preventing Staphylococcus aureus    sepsis through the inhibition of its agglutination in blood. PLoS    pathogens 2011, 7.-   10. Stearns-Kurosawa, D. J.; Osuchowski, M. F.; Valentine, C.;    Kurosawa, S.; Remick, D. G. The pathogenesis of sepsis. Annu Rev    Pathol-Mech 2011, 6, 19-48.-   11. Rivera, J.; Vannakambadi, G.; Hook, M.; Speziale, P.    Fibrinogen-binding proteins of gram-positive bacteria. Thrombosis    and haemostasis 2007, 98, 503-511.-   12. Bhattacharva, S.; Ploplis, V. A.; Castellino, F. J. Bacterial    plasminogen receptors utilize host plasminogen system for effective    invasion and dissemination. Journal of biomedicine & biotechnology    2012, 2012, 482096.-   13. Vitiello, M.; Galdiero, S.; D'Isanto, M.; D'Amico, M.; Di    Filippo, C.; Cantisani, M. Galdiero, M.; Pedone, C.    Pathophysiological changes of gram-negative bacterial infection can    be reproduced by a synthetic peptide mimicking loop 17 sequence of    Haemophilus influenzae porin. Microbes and infection/Institut    Pasteur 2008, 10, 657-663.-   14. Lahteenmaki, K.; Kuusela, P.; Korhonen, T. K. Bacterial    plasminogen activators and receptors. FEMS microbiology reviews    2001, 25, 531-552.-   15. van der Poll. T.; Herwald, H. The coagulation system and its    function in early immune defense. Thrombosis and haemostasis 2014,    112.-   16. Aktories, K.; Barbieri, J. T. Bacterial cytotoxins: Targeting    eukaryotic switches. Nature reviews. Microbiology 2005, 3, 397-410.-   17. Aktories, K.; Just, I. Clostridial rho-inhibiting protein    toxins. Current topics in microbiology and immunology 2005, 291,    113-145.-   18. Aktories, K.; Schmidt, G. A new turn in rho gtpase activation by    Escherichia coli cytotoxic necrotizing factors. Trends in    microbiology 2003, 11, 152-155.-   19. Bokoch. G. M. Regulation of innate immunity by rho gtpases.    Trends Cell Biol 2005, 15, 163-171.-   20. Boquet, P.; Lemichez, E. Bacterial virulence factors targeting    rho gtpases: Parasitism or symbiosis? Trends Cell Biol 2003, 13,    238-246.-   21. Cherfils, J.; Zeghouf. M. Regulation of small gtpases by gefs,    gaps, and gdis. Physiological reviews 2013, 93, 269-309.-   22. Lemichez. E.; Aktories, K. Hijacking of rho gtpases during    bacterial infection. Experimental cell research 2013, 319,    2329-2336.-   23. Bondu-Hawkins, V.; Schrader, R.; Gawinowicz, M. A.; Mcguire, P.;    Lawrence, D.; Hjelle, B.; Buranda, T. Elevated plasma cytokine,    thrombin, and pai-1 levels in patients with severe hantavirus    cardiopulmonary syndrome due to sin nombre virus Viruses 2014, in    press.-   24. Buranda, T.; Basuray, S.; Swanson. S.; Bondu-Hawkins. V.; Agola,    J.; Wandinger-Ness, A. Rapid parallel flow cytometry assays of    active gtpases using effector beads. Analytical biochemistry 2013,    144, 149-157.-   25. Buranda, T.; Swanson. S.; Bondu, V.; Schaefer. L.; Maclean, J.;    Mo, Z. Z.; Wycoff, K.; Belle, A. Hjelle, B. Equilibrium and kinetics    of sin nombre hantavirus binding at daf/cd55 functionalized bead    surfaces. Vinses-Basel 2014, 6, 1091-1111.-   26. Osuchowski, M. F.; Welch, K.; Siddiqui, J.; Remick, D. G.    Circulating cytokine/inhibitor profiles reshape the understanding of    the sirs/cars continuum in sepsis and predict mortality. J Immunol    2006, 177, 1967-1974.-   27. Lvovschi. V.; Arnaud, L.; Parizot, C.; Freund, Y.; Juillien, G.;    Ghillani-Dalbin, P.; Bouberima, M.; Larsen. M.; Riou, B.; Gorochov,    G.; Hausfater, P. Cytokine profiles in sepsis have limited relevance    for stratifying patients in the emergency department: A prospective    observational study. PloS one 2011, 6, e28870.

In one embodiment, our invention provides a method of diagnosing sepsisor the prognosis of the severity of viral hemorrhagic fever (“VHF”, e.g.hanviruses (hantavirus), ebola, Marburg, Lassa, and Crimean-Congohaemorrhagic fever, etc) infection in a subject, the method comprisingthe steps of:

(a) detecting the onset of sepsis and the severity of VHFinfection-associated-GTPase biomarkers in a sample (preferably a plasmasample) obtained from the subject, wherein detecting comprisescontacting the sample with a set of reagents (in preferred aspects,antibodies) which specifically bind to the sepsis associated-GTPasebiomarkers;(b) determining the presence and levels of at least one of the set ofsepsis associated-GTPase biomarkers using flow cytometry or animmunoassay selected from the group consisting of ELISA, RIA, Westernblot, luminescent immunoassay and fluorescent immunoassay; and(c) using the determined presence and levels of sepsis associated-GTPasebiomarkers to diagnose sepsis or the severity of VHF infection in theindividual.

In one embodiment, the level of at least one of the set of sepsis andVHF infection-associated-GTPase biomarkers is determined using a flowcytometry assay, the sample consists of cell sates derived from thesubject's plasma, and the flow cytometry assay comprises incubatingGTPase effector coated beads with the cell lysates to detect those celllysates that contain active, GTP-bound Ras, Rho, Ran, Arf and RabGTPases, the GTPases being selectively recruited to beads that beartheir cognate effector and being detected directly using fluorophoreconjugated monoclonal antibodies specific for each GTPase or indirectlyusing secondary antibodies with fluorophore tags.

In certain embodiments, the flow cytometry assay is a multiplex flowcytometry assay in which distinct effectors are immobilized on beads ofgraded fluorescence intensities of a fluorophore with a fixed color(wavelength) and an extra set of effector free beads is used as acontrol for nonspecific binding.

In certain embodiments of the invention, multiplexed flow cytometrydetects the presence and levels of sepsis and virus (e.g., hemorrhagicfever virus such as hanviruses (hantavirus), ebola. Marburg, Lassa andCrimean-Congo haemorrhagic fever, etc.) infection-associated-GTPasebiomarkers using:

(a) a population of beads which have two or more sizes, which arelabeled with a first fluorophore having a single wavelength (color) anda plurality of intensity levels and which are coupled to a plurality ofeffector proteins which bind to a plurality of cognate,infection-associated-GTPases(b) GTPase-specific antibodies which bind to effectorprotein-infection-associated-GTPase conjugates formed on the beads; and(c) detector antibodies which are specific to the GTPase-specificantibodies and which are labeled with a second fluorophore having awavelength (color) which is different from that of the firstfluorophore.

In certain embodiments of the invention:

(a) the effector proteins are selected from the group consisting ofPAK-1 RBD (a Rac1 and Cdc42 effector), Raf-1 RBD, RalGDS, Rg1, Rgr Rlf,α,β,γ PI (3) kinases, AF6_RA1, AF6_RA2, Nore1(Ras effector),Rhotekin-RBD (a Rho effector), RaGDS-RBD (a RAP1 effector protein),RILP-RBD (a Rab-7 effector protein), RAIN, RalGEF, RASSF1, RIN1, RIN2,PDZGEF, Tiam1, Epac1, Repac1, PLCϵ, scCYR1, spByr2 and Krit1; and(b) the first flurophore is a Rhodamine dye (preferably a redflurophore, e.g. Rhodamine Red-X) and the second fluorophore is a greenflurophore (e.g. Alexa® 488).

In another preferred embodiment, the invention provides a method ofusing a multiplexed flow cytometric assay to diagnose sepsis or a virusinfection in a subject, the method comprising:

(a) incubating a sample obtained from the subject with a population ofbeads which have two or more sizes, which are labeled with a firstfluorophore having a single wavelength (color) and a plurality ofintensity levels and which are coupled to a plurality of effectorproteins which bind to a plurality of cognate,infection-associated-GTPases;(b) incubating the beads with primary GTPase-specific antibodies;(c) mixing the incubated fluorescent beads of step (b) with secondaryantibodies which are specific to primary GTPase-specific antibodies andwhich are labeled with a second fluorophore having a wavelength (color)which is different from that of the first fluorophore; and(d) measuring the fluorescence intensity of the mixed beads of step (c)using flow cytometry to determine the presence and level ofinfection-associated-GTPase in the sample.

In certain embodiments, multiplexed assays used in methods of theinvention can be conducted at speed of about 40 wells per minute and theassay can process at least 96 or 384 well-plates in a time period ofbetween about 2 to about 15 minutes, more preferably in about 3 to about12 minutes.

In certain embodiments, a subject's sepsis or virus infection diagnosisincludes the additional steps of:

(a) analyzing the impairment of hemostasis and/or bacterialinfection-related increase in thrombogenesis and fibrinolysis bymeasuring thrombin and/or plasmin levels in a blood sample obtained fromthe subject; and

(b) comparing measured thrombin and/or plasmin levels to controlthrombin and/or plasmin levels determined in a healthy control subject;

wherein measured thrombin and/or plasmin levels which exceedcorresponding control levels are indicative of the onset or progressionof sepsis or virus infection.

Analyses of hemostasic and/or bacterial infection-related increase inthrombogenesis and fibrinolysis can be conducted in parallel withmultiplexed flow cytometry assays described herein. An electriccell-substrate impedance sensing (ECIS) cell-based assay can be used toascertain thrombin and/or plasmin-associated disruption of cellularfunction in cells exposed to a sample of the subject's plasma. See, forexample, Bondu, et al, Viruses, 7, 559-589 (2015), relevant portions ofwhich are incorporated by reference herein. Preferably, ECIS cells areplated at confluence in electrode-containing dishes, cellular impedanceis measured continuously at a single frequency, increasing cell barrierfunction is confirmed by increasing resistance, cells are exposed to asample of the subject's plasma and any decrease in cell monolayerresistance is correlated to sepsis-associated thrombin and/orplasmin-associated disruption of cellular function.

One or more steps of the novel diagnostic methods described herein canbe conducted in a high-throughput fashion and/or can be done in silico.

One embodiment of the invention diagnoses the degree of severity of VHFinfection.

The active GTPase effector trap flow cytometry assay (G-trap) describedand claimed herein has significant advantages over standard pulldowntechniques, including: (1) the fact that G-trap enables rapidmeasurement, sensitivity and analysis with results within four hours,compared to days required for other assays; (2) G-trap uses smallsamples e.g. <100,000 cells grown in a 48 well plate, well below therequisite minimum of 2×10⁶ cells for single ELISA based measures, (3)G-trap can measure multiple GTPases from a single lysate using amultiplex approach (FIG. 11A), whereas conventional assays require theentire lysate for measurement of a single GTPase, and (4) G-trapfacilitates replicate measures on scarce cell samples.

Thus, diagnostic methods of the invention improve clinical care byidentifying a manageable basis set of response factors in the patient'sblood that can be monitored in real time in order to enable clinicalintervention during a limited window of effective therapy.

The diagnostic methods of the invention are also useful to monitortherapeutic effect of intervention and prognosis for survival.

These and other aspects of the invention are described further in theDetailed Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: a, b, c, d, e, f shows that UV-killed SNV^(R18) induces vigorousremodeling of the actin cytoskeleton of Vero E6 cells, causing loss ofcell adhesion, a. Confocal microscopy images of resting Vero E6 cellsmonolayers transiently transfected with the cell adhesion marker,paxillin GFP. Individual cells are delineated with lines. Cellsdesignated 1 and 2 are also defined by their surface area. The whitesquare was included as size reference for cell 1, b. Time lapse frametaken 42 min after cells were exposed to 10,000 SNV^(R18)particles/cell. SNV^(R18) induces remodeling of the actin cytoskeletonof Vero E6 cells. Cells designated 1 and 2 and surrounding cells shrankin size and experience loss of cell adhesion to each other and thecoverslip. c. Resting Vero E6 cells stained showing actin stained withAlexa Fluor 488® phalloidin. d. RhoA induced F-actin stress fibers areshown 15 min after SNV exposure, e. EFCB assay for virus induced GTPRac1 and GTP RhoAGTP. Vero E6 cells were serum starved for 24 hours andactivated with 10,000 SNV^(R18)/cell. Rac1 and RhoA were measured onPAK1 and Rhotekin beads at 3 and 20 min after virus exposure. The errorsrepresent standard deviations of 3 separate measurements, f. Vero E6cells were stimulated with: calpeptin (calpptn) to activate RhoA, EGF toactivate Rac1 and RhoA, NSC23766 (NSC) to suppress Rac1 activity.Control samples (rest) were mock-treated with 0.1% DMSO to account forcompound solvent. The errors represent standard deviation of 3independent experiments measured in duplicate at a time.

FIG. 2 shows that Rap1 and H-Ras are sequentially activated by SNV^(R18)in Vero E6 cells. Vero E6 cells were serum starved for 24 hours andactivated with 10,000 SNV^(R18)/cell. Rap and H-Ras were measured on Raland Raf-functionalized beads at 3 and 20 min after virus exposure. Cellswere treated with FTI277 a specific inhibitor of H-Ras and 8-Cpt-2m-cAMP(cAMP) an activator of Rap1. Inhibition of H-Ras induced a near 2-foldincrease active Rap1, while activation of Rap1 decreased basal levels ofH-Ras. In virus-activated cells. Rap1 and H-Ras were activatedsequentially. Rap 1 was activated within the first 3 min andsubsequently deactivated at 20 mins and H-Ras was fully active at 20min. Error bars represent standard deviations 3 separate measurements.

FIG. 3 shows that Rab7 mediate trafficking of SNV^(R18) from early tolate endosomes in Vero E6 cells. 3A shows that EGF was used todemonstrate the tractability of monitoring cellular Rab7 activationusing beads functionalized with RILP-RBD. 3B shows that the highestRab7-GTP levels were realized at 20 min after virus exposure.

FIG. 4 shows that SNV induces upregulation of GTP bound RhoGTPases inVero E6 cells. Bar graph shows GLISAs measuring kinetics ofadhesion-dependent Rho protein (Rac1 and Cdc42) activation.PI-PLC-treated cells were cleared of DAF surface expression, a receptorfor virus binding. Serum starved cells were stimulated with SNVparticles as described in the methods and compared with unstimulatedcells (0 min). Control cells were mock-treated with media to account forvirus diluent. After cell lysis, the amounts of active Rac1 and RhoAwere quantified based on p21-activated protein kinase or Rhotekinbinding by GLISA respectively. The error bars represent the standarddeviation of three independent measurements.

FIGS. 5: A and B shows endogenous Rab7-positive endosomes were stainedwith antiRab7 antibodies. SNV^(R18) particles were allowed to bind tocells at 15° C. for 30 minutes, washed, and fixed at various times afterthe temperature was quickly raised to 37° C. At 37° C. the cargo wasallowed to synchronously move towards perinuclear space. The degree ofcolocalization of Rab7 and SNV^(R18) was then measured at each timepoint. Colocalization was assessed using Slidebook software as shown inthe plot in FIG. 5B. The experiments were done in duplicate, andcolocalization was analyzed for at least 10 cells in each image.

FIG. 6. ECIS measurement of barrier dysregulation caused by patientplasma in Vero E6 cells. Plot of normalized resistance versus Timeshowing the effects of trauma patient plasma (for days 0, 1, 3 and 5) oncell barrier function over the course of 10 hrs.

FIGS. 7: A and B show treatment of septic (#P013) and aseptic patient(#P019)-plasma samples with inhibitors of thrombin (Argatroban) andplasmin (Tranexamic acid) display differential ECIS profiles. Dottedlines are healthy controls. A. Samples treated with argatroban andtranexamic acid recover monolayer integrity after a few hours albeit therecovery is slower in P013 patient from the start. B. Monolayers exposedto plasma from a P013 are partially refractory to argatroban onlytreatment, whereas cells from P019 are responsive to argatroban only.

FIGS. 8: A, B and C show ECIS profile of cell barrier function in cellmonolayers treated with serially collected plasma of aseptic (p19) andseptic (p13) trauma patients. Septic patient plasma induces significantloss of cell barrier function compared to sterile inflammation. B. Plotof active RhoA induced in cell monolayers by p13 and p19 plasma versusday of sample collection. Day −1 refers to healthy control plasma.Decline in RhoA activity after day 4 coincides with antibiotictreatment. C. Plot of RhoA activity induced by samples treated withArgatroban (thrombin inhibitor) and tranexamic acid (plasmin inhibitor).Data suggest that the RhoA activity is due to elevated thrombin andplasmin in serial samples from p13. Rap1 activation (not shown) wasrefractory to argatroban treatment, suggesting activity of otherinflammatory mediators.

FIG. 9. Schematic model Gtrap Assay: 9A. Multiplex assay based on 12sets of cytoplex beads encoded with graded levels of red fluorescence(700 nm). 9B shows calibration fluorescence histograms of GST-GFPnon-specific and specific binding. Histograms show that site occupancyis uniform among the 12-plex beads. L1-L12 refers to the intensity levelassociated with beads functionalized with distinct effectors.

FIGS. 10. A and B show multiplex measurements of GTPases activated incell monolayers exposed of serial plasma samples from septic patientsP14 and P18. A. Plot of fold increase in the activity of RhoA, Rap1 andRac1 in cell monolayers exposed to P14 plasma for 30 min. Day −1 refersto healthy control plasma. P14 was admitted after a low speed car crushwith minor injuries. Suspected of community-acquired pneumonia (S.pneumonia from sputum culture on admission). B. Plot of RhoA and Rap1activity in cells exposed P18 plasma samples for 30 min. P18 was runover by a car and sustained serious injuries. He was admitted to theintensive care unit for mechanical ventilation, further resuscitation,and insertion of arterial and central venous lines. In line sepsisbacteremia, diagnosed with bacterial infection on day 9 (Staph aureus)and 11 (Staph epidermis).

FIGS. 11. A, B, C, and D show the GTPase effector trap flow cytometryassay (G-trap). A. Plot of red fluorescence (FL4) versus sidescatter(SSC) of a set 12 of Cyto-Plex™ beads dyed with 12 discrete levels(L_(i) i=1, . . . 12) of 700 nm fluorescence. B. Li beads are coatedwith effectors for GTPase bait, some beads (e.g. L4) are notfunctionalized with effectors and used as controls for non-specificbinding. L_(i) GSH beads individually coated with specific effectors fordetection of discrete active GTPase populations are added to a sampletube containing cell lysates. As illustrated GTP-bound GTPases (P³ inschema; P²=GDP) are recruited to their cognate effectors immobilized onbeads, while GDP-bound GTPases remain unbound. Bead-bound active GTPaseassemblies are quantified using fluorescently labeled antibodies. In theG-trap assay, the letters a, b, c, d, and e identify and link effectorsto their cognate GTPases and fluorescently labeled reporter antibodies(FL1 (520 nm emission). C. Prototypical multiplex format, electronicgates on red fluorescence (FL4) versus side scatter (SSC) on the flowcytometer are used to select individual L, beads that bear knownanalytes, after which bead associated green fluorescence reflectingbound antibody (FL1) in each gate were measured. D. Reporter antibodycross talk was examined to determine the significance of differentialnon-specific staining of cytoplex beads. Multiplex samples were preparedby exposing GSH beads to multiple antibodies and measuring the aggregatefluorescence of nonspecifically bound antibodies. Fraction samples wereprepared by staining, washing and measuring fluorescence of beadpopulations separately. Mixed fraction samples were prepared as thefraction samples but mixed and measured in multiplex format.

FIG. 12. A, B, C, D show G-trap Assay Validation. Serum starved Helacells were stimulated with: 10 nM EGF to activate Rac1, Cdc42, and RhoA,100 μM NSC23766 to suppress Rac1 activity, 10 μM ML141 to inhibit Cdc42,1 μM Calpeptin (clptn) to activate RhoA. Control samples (rest andnon-cognate GSH effector beads) were mock-treated with 0.1% DMSO toaccount for compound solvent. A. Nonspecific binding-corrected plot offold change (relative to resting cells) in median channel fluorescence(MCF) flow cytometer readings corresponding to changes in active Rac1and RhoA in cells treated with EGF as indicated in panel. NSC refers toresting cells treated with NSC23766. EGF-NSC refers to cells treatedwith NSC23766 (1 h) and then EGF stimulated (3 min) before analysis ofcell lysates for activated GTPases by flow cytometry. B. Data werecollected after 20 min of EGF stimulation where applicable, all otherconditions are the same as Panel A. C. Conditions as in Panel Ameasuring changes in active Cdc42 and RhoA. ML 141 or CID2950007 servedas the Cdc42 specific inhibitor. D. Conditions as in Panel B withapplicable changes for Cdc42. The errors represent standard deviation of3 independent experiments measured in duplicate each time. **P<0.001 forall data compared to resting (rest) cells.

FIGS. 13. A, B, C, D, E, F, G, H, and I show UV-killed SNV^(R18) inducesvigorous remodeling of the actin cytoskeleton of Vero E6 cells, causingloss of cell adhesion. Confocal microscopy images of resting Vero E6monolayers transiently transfected with the cell adhesion marker,paxillin GFP. Individual cells are outlined. A. Cells designated 1 and 2before virus exposure are also defined by their surface area. The whitesquare was included as size reference for cell I. B. After 15 minexposure to 10,000 SNV^(R18) particles/cell the size of reference cell 1is decreased relative to the rectangular marker. C. Time lapse frametaken 42 min after cells were exposed SNV^(R18) shows how cells 1 and 2shrank in size and lost cell adhesion to each other and the coverslip.Arrows mark broken cell-cell junctions. D. Resting Vero E6 cells stainedfor actin with Alexa Fluor 488®phalloidin. E. RhoA induced F-actinstress fibers are shown 15 min activation with calpeptin and F, afterSNV treatment. G. Resting Vero cells stained with integrin affinitysensitive AP-5 antibodies. H. Increased AP-5 staining in Mn²⁺ activatedcells. I. Detection of differential activation of β₃ integrins at theperinuclear and peripheral regions of Vero cells using AP5 antibodies.Arrows mark broken cell-cell junctions.

FIGS. 14. A and B show SNV induces the activity of several GTPases inVero E6 cells. A. Vero E6 cells were serum starved for 24 h and treatedwith 10,000 SNV^(R18)/cell. Active Rac1 and RhoA were detected in celllysates at 3 min and 20 min after virus exposure using PAK1 and Rhotekinbeads, respectively. The errors represent standard deviations in 3separate measurements. B. Rap1 and H-Ras were sequentially activated bySNV^(R18) in Vero E6 cells. Vero E6 cells were serum starved for 24 hand treated with 10,000 SNV^(R18)/cell. Active Rap1 and H-Ras weremeasured on Ral and Raf-functionalized beads, respectively, at 3 and 20min after virus exposure. Cells were treated with the cAMP analog8-pCPT-2′-O-methyl-cAMP (O-Me-cAMP), which stimulates the Epac/Rap1pathway as a specificity control. Error bars represent standarddeviations 3 separate measurements.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used throughout the specification todescribe the present invention. Where a term is not specifically definedherein, that term shall be understood to be used in a manner consistentwith its use by those of ordinary skill in the art.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention. In instanceswhere a substituent is a possibility in one or more Markush groups, itis understood that only those substituents which form stable bonds areto be used.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow.

“Sepsis and/or viral hemorrhagic fever-associated GTPases” include butare not limited to RhoA, Rac1, and Cdc42 and the isoforms thereof.

“Sepsis-associated” and “Viral hemorrhagic fever associated” GTPasebiomarkers” are described hereinafter. Non-limiting examples of sepsisand Viral hemorrhagic fever-associated GTPase biomarkers include PAK-1RBD-Rac1 conjugates and PAK-1 RBD-Cdc42 conjugates, Raf-1 RBD-Rasconjugates, Rhotekin-RBD-Rho conjugates. RaGDS-RBD-RAP1 conjugates andRILP-RBD-Rab-7 conjugates, among others.

“GTPase effectors” are molecules which bind to GTPases and effect theirfunction. GTPase effectors for use in the present invention includePAK-1 RBD (a Rac1 and Cdc42 effector), Raf-1 RBD, RalGDS, Rgl, Rgr Rlf,α,β,γ PI (3) kinases, AF6_RA1, AF6_RA2, Nore1(Ras effector),Rhotekin-RBD (a Rho effector) RalGDS-RBD (a RAP1 effector protein).RILP-RBD (a Rab-7 effector protein), RAIN, RalGEF, RASSF1, RIN1, RIN2,PDZGEF, Tiam1, Epac1, Repac1, PLCϵ, scCYR1, spByr2 and Krit1, amongnumerous others.

The GTPase RhoA effectors include:

Cit, Cnksr1, Diaph1, Diaph2, DgkQ, FInA, KcnA2, Ktn1, Rtkn1, Rtkn2,Rhpn1, Rhpn2, Itprl1, PlcG1, PI-5-p5K, Pld1, Pkn1, Pkn2, Rock1, Rock2,PrkcA and Ppp1r12A.

The GTPase Rac1 effectors include Sra1, IRSp53, PAK1, PAK2 and PAK3.

The GTPase Cdc42 effectors include Wiskott-Aldrich syndrome protein,N-WASP, IRSp53, Dia2, Dia3, ROCK1 and ROCK2.

In a preferred embodiment, effector proteins are selected from the groupconsisting of PAK-1 RBD (a Rac1 and Cdc42 effector), Raf-1 RBD, (a Raseffector). Rhotekin-RBD (a Rho effector), RalGDS-RBD (a RAP1 effectorprotein) and RILP-RBD (a Rab-7 effector protein).

Glutathione (GSH), a ubiquitous tripeptide, is an important cellularconstituent, and measurement of reduced and oxidized glutathione is ameasure of the redox state of cells. Glutathione-S-transferase (GST)fusion proteins bind naturally to beads derivatized with glutathione,and elution of such bead-bound fusion proteins with buffer containingmillimolar glutathione is a commonly used method of proteinpurification. Many protein-protein interactions have been established byusing GST fusion proteins and measuring binding of fusion proteinbinding partners by GST pulldown assays. Tessema, et al.,“Glutathione-S-transferase-green fluorescent protein fusion proteinreveals slow dissociation from high site density beads and measures freeGSH”, Cytometry, 69A: 326-334. doi: 10.1002/cyto.a.20259.

As explained in Example 3 hereinafter, to simplify bacterial expressionand purification of effector proteins, it is preferable to use only theGTPase binding domains (RBD) of cognate effectors. In preferredembodiments, effector coated beads are incubated with cell lysates thatcontain active, GTP-bound Ras, Rho and Rab GTPases. The GTPases areselectively recruited to beads that bear the cognate effector and aredetected directly using fluorophore conjugated monoclonal antibodiesspecific for each GTPase or indirectly using secondary antibodies withfluorophore tags. In a non-limiting flow cytometry example explainedfurther hereinafter (see e.g. Example 3), glutathione beads canfunctionalized with effectors for Rap1 (GST RalGDS-RBD) and H-Ras (Raf-1RBD) to simultaneously assay activated Rap1 and H-Ras GTPases from celllysates. As illustrated in Example 1, GST (Glutathione S-Transferase)conjugates of various GTPase-effector proteins can be bound tosuspensions of GSH beads to in order to form molecular assembliesnecessary for capturing GTP-bound GTPases from cell lysates. Thecharacteristic kinetic and equilibrium binding constants ofglutathione-S-transferase (GST) fused to Green fluorescent protein(GFP), can be used to establish optimal stoichiometric mixtures of GSHbeads and specific GST effector fusion proteins for desired siteoccupancies of the GST effector proteins used in the flow cytometryassays.

“Reagents which specifically bind to the sepsis (or Viral hemorrhagicfever) associated-GTPase biomarkers” include GTPase biomarker proteinpurification reagents, antibodies to GTPase biomarker polypeptides orpeptides thereof, nucleic acid primers specific for genes which expressGTPase biomarkers, arrays of GTPase biomarker-related nucleic acidprobes, signal producing system reagents, etc. Useful reagents includearrays that comprise probes, e.g. arrays of antibodies or arrays ofoligonucleotides; or other reagents that may be used to detect theexpression of GTPase biomarkers. Those of ordinary skill in the art knowhow to identify and make the aforementioned reagents. See e.g. Spiegel,et al., Direct Targeting of Rab-GTPase-Effector Interactions, AngewandteChemie International Edition Volume 53, Issue 9, pages 2498-2503, Feb.24, 2014; Kahn, et al., “Structural Biology of Arf and Rab GTPases'Effector Recruitment and Specificity”, Structure 21, Aug. 6, 2013.

The basics of flow cytometry and multiplexed flow cytometry arewell-known to those of ordinary skill in the art. See e.g. the technicaldescription and supporting references cited at:http://www.einstein.yu.edu/research/facilities/facs/page.aspx?id=22632.A useful summary of certain types of multiplexed flow cytometry assaysis provided in U.S. Patent Application Document No. 20140206008 asfollows. “Luminex MultiAnalyte Profiling (xMAP) technology, previouslyknown as FlowMetrix and LabMAP (Elsha1 and McCoy, 2006), is a multiplexbead-based flow cytometric assay that is gaining recognition as a methodfor analyte quantitation. This technology utilizes 5.6-micronpolystyrene beads that are internally dyed with different intensities ofred and infrared fluorophores. Currently there are 100 beads, each witha unique spectral make up which allows the mixing of several bead setsand, in theory, enabling the detection of up to 100 different analytesper assay (Vignali, D. A. A., J Immunol Methods, 243:243-255 (2000)).The beads can be bound by various capture reagents such as antibodies,oligonucleotides, and peptides, therefore facilitating thequantification of various proteins, ligands, DNA and RNA (Fulton, R. J.et al., Clin Chem, 43:1749-1756 (1997); Kingsmore, S. F., Nat Rev DrugDiscov, 5:310-321 (2006); Nolan, J. P. and Mandy, F., Cytometry Part A,69A:318-325 (2006)). The assays are run on a 96-well plate format,followed by detection on a Luminex 100 instrument. As the beads runthrough the instrument, the internal dyes are excited by a laser whichresults in the classification of each bead. Another laser excites thereporter dye which is directly proportional to the amount of analytebound to each bead (Vignali, D. A. A., J Immunol Methods, 243:243-255(2000); Ray, C. A. et al., J Pharma Biomed Anal, 36:1037-1044 (2005)).The resulting fluorescence is recorded by the instrument which thenprovides the median fluorescence unit obtained from measuring 100 beads.Luminex xMAP technology has many applications including proteinexpression profiling, gene expression profiling, genotyping,immunodiagnostics, and genetic disease diagnostics. Although single-plexbead-based assays have been available for a long time; technologicaldevelopments have enhanced the development of multiplex bead-basedassays enabling the utilization of this method for quantitation of apanel of protein markers simultaneously (Linkov, F. et al., CancerEpidemiol Biomarkers Prey, 16:102-107 (2007); Prabhakar, U. et al., JImmunol Methods, 260:207-218 (2002)). The advantage of Luminex xMAPtechnology lies in its high sensitivity, throughput and efficiency(Vignali, D. A. A., J Immunol Methods, 243:243-255 (2000); DuPont, N. C.et al., J Reprod Immunol, 66:175-191 (2005)). Significant reduction intime and costs results from multiplexing when compared to ELISA. ELISAis more expensive and time-consuming to perform when many proteins areto be measured using many single-plex protein specific assays (de Jager,W. and Rijkers, G. T., Methods, 38:294-303 (2006)). On the contrary,many protein analytes can be measured by the multiplexed bead-basedassay with a single plate. This is extremely important for clinicalstudies where sample volumes are limited (Liu, M. Y. et al., Clin Chem,51:1102-1109 (2005)). Bead-based assay is more accurate because themedian fluorescence is obtained from the readout of at least 50 to 100beads. Thus each bead is functioning as a duplicate, making this assaymore reliable (Vignali, D. A. A., J Immunol Methods, 243:243-255 (2000);Kettman, J. R. et al., Cytometry, 33:234-243 (1998)).”

Multiplexed flow cytometric assays of the invention can use, e.g. 2, 4,8, 16, 32, 64, 128, 256, or 12, 24, 36, 48 or 60 distinct sets offluorescent spheres (beads) or microspheres and a standard benchtop flowcytometer interfaced with a personal computer containing a digitalsignal processing board and programmed with a variety of operatingsoftware. Individual sets of beads or microspheres (microbeads) can bemodified with reactive components such as antigens, antibodies, oroligonucleotides, and then mixed to form a multiplexed assay set. Thedigital signal-processing hardware and software control the flowcytometer and perform real-time data processing, allowing multipleindependent reactions to be analyzed simultaneously in qualitative andquantitative immunoassays for multiple serum proteins in both captureand competitive inhibition assay formats.

Multiplexed beads can be assigned to two or more groups which performdistinct assays and are distinguished by characteristics that enabledistinct detection of assay group results. Bead size can be adistinguisher; beads are defined by distinct sub-sizes and are groupedinto distinct sub-ranges, e.g. 2, 4, 8, 16, 32, 64, 128 sub-ranges, eachof which conducts a unique assay. Particle size sub-ranges and meandiameter spacing of adjacent sub-ranges permit differentiation of thesub-ranges. Preferred sub-ranges can vary by about +/−5% CV or less ofthe mean diameter, where CV is the coefficient of variation and isdefined as the standard deviation of the particle diameter divided bythe mean particle diameter times 100 percent. Minimum spacing betweenmean diameters among the various sub-ranges can depend on bead sizedistribution and flow cytometry sensitivity. Fluorescencedifferentiation is achieved by using various fluorescent materials inthe beads having different fluorescent emission spectra. Fluorescencecan distinguish sub-groups and serve as an assay detector.

“A gate in cytometry is a set of value limits (boundaries) that serve toisolate a specific group of cytometric events from a large set. Gatescan be defined by discrimination analysis, or can simply be drawn arounda given set of data points on a print-out and then converted to acomputer-useful form. Gates can be implemented with a physical blinder.Gates may be used either to selectively gather data or to segregate datafor analysis. Gates are divided mathematically into inclusive gates andexclusive gates. Inclusive gates select data that falls within thelimits set, while exclusive gates select data that falls outside thelimits. A live gate is a term used for a process that prevents theacquisition by the computer of non-selected data from the flowcytometer. (see, for example, Osborne, G. W. (2000) “Regions and Gates”Flow Cytometry Software Workshop: 2000, page 3).” See U.S. PatentApplication Document No. 20120083007.

Other types of assays besides flow cytometric assays (e.g. ELISA, RIA,Western blot, luminescent immunoassay and fluorescent immunoassay) canbe used to measure the amount of binding between said protein moleculeand an anti-protein antibody by the use of enzymatic, chromodynamic,radioactive, magnetic, or luminescent labels which are attached toeither the anti-protein antibody or a secondary antibody which binds theanti-protein antibody. In addition, other high affinity ligands may beused. Immunoassays which can be used include e.g. ELISAs, Western blotsand other techniques known to those of ordinary skill in the art (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999 and Edwards R,Immunodiagnostics: A Practical Approach, Oxford University Press,Oxford; England, 1999). All these detection techniques may also beemployed in the format of microarrays, protein-arrays, antibodymicroarrays, tissue microarrays, electronic biochip or protein-chipbased technologies (see Schena M., Microarray Biochip Technology, EatonPublishing, Natick, Mass., 2000).

Certain diagnostic and screening methods of the present inventionutilize an antibody, preferably, a monocolonal antibody, capable ofspecifically binding to a protein as described herein or activefragments thereof. The method of utilizing an antibody to measure thelevels of protein allows for non-invasive diagnosis of the pathologicalstates of sepsis or Viral hemorrhagic fever infections. In a preferredembodiment of the present invention, the antibody is human or ishumanized. The preferred antibodies may be used, for example, instandard radioimmunoassays or enzyme-linked immunosorbent assays orother assays which utilize antibodies for measurement of levels ofprotein in sample. In a particular embodiment, the antibodies of thepresent invention are used to detect and to measure the levels ofprotein present in a plasma sample.

Humanized antibodies are antibodies, or antibody fragments, that havethe same binding specificity as a parent antibody, (i.e., typically ofmouse origin) and increased human characteristics. Humanized antibodiesmay be obtained, for example, by chain shuffling or by using phagedisplay technology. For example, a polypeptide comprising a heavy orlight chain variable domain of a non-human antibody specific for adisease related protein is combined with a repertoire of humancomplementary (light or heavy) chain variable domains. Hybrid pairingsspecific for the antigen of interest are selected. Human chains from theselected pairings may then be combined with a repertoire of humancomplementary variable domains (heavy or light) and humanized antibodypolypeptide dimers can be selected for binding specificity for anantigen. Techniques described for generation of humanized antibodiesthat can be used in the method of the present invention are disclosedin, for example, U.S. Pat. Nos. 5,565,332; 5,585,089; 5,694,761; and5,693,762. Furthermore, techniques described for the production of humanantibodies in transgenic mice are described in, for example, U.S. Pat.Nos. 5,545,806 and 5,569,825.

Antibodies or antibody fragments employed in such screening tests may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The blocking or reduction of biologicalactivity or the formation of binding complexes between thedisease-related protein and the agent being tested can be measured bymethods available in the art.

Other techniques for drug screening which provide for a high throughputscreening of compounds having suitable binding affinity to a protein, orto another target polypeptide useful in modulating, regulating, orinhibiting the expression and/or activity of a disease, are known in theart. For example, microarrays carrying test compounds can be prepared,used, and analyzed using methods available in the art. See, e.g.,Shalon, D. et al., 1995, International Publication No. WO95/35505,Baldeschweiler et al., 1995, International Publication No. WO95/251116;Brennan et al., 1995, U.S. Pat. No. 5,474,796; Heller et al., 1997, U.S.Pat. No. 5,605,662.

Other screening techniques, which can also serve to determine thepresence and levels of sepsis associated-GTPase biomarkers arewell-known to those or ordinary skill in the art. See, e.g., Enna etal., eds., 1998, Current Protocols in Pharmacology, John Wiley & Sons,Inc., New York N.Y. Assays will typically provide for detectable signalsassociated with the binding of the compound to a protein or cellulartarget. Binding can be detected by, for example, fluorophores, enzymeconjugates, and other detectable labels well known in the art. Theresults may be qualitative or quantitative.

To determine specific binding, various immunoassays may be employed fordetecting, for example, human or primate antibodies bound to the cells.Thus, one may use labeled anti-hIg, e.g., anti-hIgM, hIgG orcombinations thereof to detect specifically bound human antibody.Various labels can be used such as radioisotopes, enzymes, fluorescers,chemiluminescers, particles, etc. There are numerous commerciallyavailable kits providing labeled anti-hIg, which may be employed inaccordance with the manufacturer's protocol.

In one embodiment, a kit can comprise; (a) at least one reagent which isselected from the group consisting of (i) reagents that detect atranscription product of the gene coding for a protein marker asdescribed herein (ii) reagents that detect a translation product of thegene coding for proteins, and/or reagents that detect a fragment orderivative or variant of said transcription or translation product; (b)instructions for diagnosing, or prognosticating a disease, ordetermining the propensity or predisposition of a subject to developsuch a disease or of monitoring the effect of a treatment by determininga level, or an activity, or both said level and said activity, and/orexpression of said transcription product and/or said translation productand/or of fragments, derivatives or variants of the foregoing, in asample obtained from said subject; and comparing said level and/or saidactivity and/or expression of said transcription product and/or saidtranslation product and/or fragments, derivatives or variants thereof toa reference value representing a known disease status (patient) and/orto a reference value representing a known health status (control) and/orto a reference value; and analyzing whether said level and/or saidactivity and/or expression is varied compared to a reference valuerepresenting a known health status, and/or is similar or equal to areference value representing a known disease status or a referencevalue; and diagnosing or prognosticating a disease, or determining thepropensity or predisposition of said subject to develop such a disease,wherein a varied or altered level, expression or activity, or both saidlevel and said activity, of said transcription product and/or saidtranslation product and/or said fragments, derivatives or variantsthereof compared to a reference value representing a known health status(control) and/or wherein a level, or activity, or both said level andsaid activity, of said transcription product and/or said translationproduct and/or said fragments, derivatives or variants thereof issimilar or equal to a reference value and/or to a reference valuerepresenting a known disease stage, indicates a diagnosis or prognosisof a disease, or an increased propensity or predisposition of developingsuch a disease, a high risk of developing signs and symptoms of adisease.

Reagents that selectively detect a transcription product and/or atranslation product of the gene coding for proteins can be sequences ofvarious length, fragments of sequences, antibodies, aptamers, siRNA,microRNA, and ribozymes. Such reagents may be used also to detectfragments, derivatives or variants thereof.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal, especiallyincluding a domesticated animal and preferably a human, to whom atreatment, including prophylactic treatment (prophylaxis) is provided.For treatment of those infections, conditions or disease states whichare specific for a specific animal such as a human patient, the termpatient refers to that specific animal. In most instances, the patientor subject is a human patient of either or both genders.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or component which, when used withinthe context of its use, produces or effects an intended result, whetherthat result relates to the prophylaxis and/or therapy of an infectionand/or disease state or as otherwise described herein. The termeffective subsumes all other effective amount or effective concentrationterms (including the term “therapeutically effective”) which areotherwise described or used in the present application.

Sepsis is a clinical syndrome that complicates severe infection. It ischaracterized by the cardinal signs of inflammation (vasodilation,leukocyte accumulation, increased microvascular permeability) occurringin tissues that are remote from the infection. Systemic inflammatoryresponse syndrome (SIRS) is an identical clinical syndrome thatcomplicates a noninfectious insult (e.g., acute pancreatitis, pulmonarycontusion). Current theories about the onset and progression of sepsisand SIRS focus on dysregulation of the inflammatory response, includingthe possibility that a massive and uncontrolled release ofproinflammatory mediators initiates a chain of events that lead towidespread tissue injury. This response can lead to multiple organdysfunction syndrome (MODS), which is the cause of the high mortalityassociated with these syndromes.

Sepsis is typically associated with a bacterial infection and ischaracterized by a whole-body inflammatory state (SIRS) and the presenceof a known or suspected infection. The body may develop thisinflammatory response by the immune system to bacteria presence in theblood, urine, lungs, skin, or other tissues. Sepsis is also referred toas “blood poisoning” or septicemia. Severe sepsis is the systemicinflammatory response, plus infection, plus the presence of at least oneorgan dysfunction. Septicemia (also sometimes referred to as bacteremia)refers to the presence of pathogenic organisms in the bloodstream,leading to sepsis.

An S. aureus infection can cause septic arthritis. Bacterial arthritis(or septic arthritis) is a rapidly progressive and highly destructivejoint disease in humans. Clinical symptoms of septic arthritis includered, swollen, warm, painful and dysfunctional joints. Septic arthritisdevelops when bacteria spread through the bloodstream to a joint and itmay also occur when the joint is directly infected with a microorganismfrom an injury or during surgery. The most common sites for this type ofinfection are the knee and hip.

In the United States, sepsis is the second-leading cause of death innon-coronary ICU patients, and the tenth-most-common cause of deathoverall according to data from the Centers for Disease Control andPrevention (the first being heart disease). Sepsis is common and alsomore dangerous in elderly, immunocompromised, and critically illpatients. It occurs in 1-2% of all hospitalizations and accounts for asmuch as 25% of intensive-care unit (ICU) bed utilization. It is a majorcause of death in intensive-care units worldwide, with mortality ratesthat range from 20% for sepsis to 40% for severe sepsis to >60% forseptic shock.

Septic shock is a medical emergency caused by decreased tissue perfusionand oxygen delivery as a result of severe infection and sepsis, thoughthe microbe may be systemic or localized to a particular site. It cancause multiple organ dysfunction syndrome (formerly known as multipleorgan failure) and death. Its most common victims are children,immunocompromised individuals, and the elderly, as their immune systemscannot deal with the infection as effectively as those of healthyadults. Frequently, patients suffering from septic shock are cared forin intensive care units. The mortality rate from septic shock isapproximately 25%-50%. See United States Patent Application Document No.20140162978.

Adequate management of septic patients is often complicated by delay inadministering therapy after sepsis has been recognized. Every hour delayin the administration of appropriate antibiotic therapy there isassociated with a significant rise in mortality.

“Sepsis” as used herein includes all of the aforementioned septicstates, conditions and clinical symptoms, e.g. “sepsis” includes but isnot limited to systemic inflammatory response syndrome (SIRS),septicemia, septic arthritis and septic shock.

Hantaviruses belong to the bunyavirus family of viruses. There are fivegenera within the family: bunyavirus, phlebovirus, nairovirus,tospovirus, and hantavirus. Each is made up of negative-sensed,single-stranded RNA viruses. All these genera include arthropod-borneviruses, with the exception of hantavirus, which is rodent-borne. Likeother members of the bunyavirus family, hantaviruses are envelopedviruses with a genome that consists of three single-stranded RNAsegments designated S (small), M (medium), and L (large). All hantaviralgenes are encoded in the negative (genome complementary) sense. The SRNA encodes the nucleocapsid (N) protein. The M RNA encodes apolyprotein that is cotranslationally cleaved to yield the envelopeglycoproteins G1 and G2. The L RNA encodes the L protein, whichfunctions as the viral transcriptase/replicase. Within virions, thegenomic RNAs of hantaviruses are thought to complex with the N proteinto form helical nucleocapsids, which circularize due to sequencecomplementarity between the 5′ and 3′ terminal sequences of each genomicsegment.

Sin Nombre virus (SNV), a hantavirus, was first isolated from rodentscollected on the premises of one of the initial HPS patients in the FourCorners region. Isolation was achieved through blind passage inPeromyscus maniculatus and subsequent adaptation to growth in Vero E6cells. Additional viral strains have also been isolated from P.maniculatus associated with a fatal case in California and P. leucopusfrom the vicinity of probable infection of a New York case. Black CreekCanal virus was isolated from S. hispidus collected near the residenceof a human case in Dade County, Florida.

Several members of the hantavirus genus cause different forms ofhemorrhagic fever with renal syndrome (HFRS), an ancient disease firstdescribed in Russia in 1913. The four viruses that are associated withHFRS, each named for the region from where they were first isolated,have different primary rodent hosts: Apodemus agrarius (the stripedfield mouse) for Hantaan virus, Rattus norvegicus (the Norway rat) andRattus rattus (the black rat) for Seoul virus, Clethrionomys glareolus(the bank vole) for Puumala virus, and Apodemus flavicollis (theyellow-necked field mouse) for Dobrava virus. Hantaan virus from Koreaand Dobrava virus from Slovenia are associated with a severe form ofHFRS characterized by renal failure that can precede pulmonary edema anddisseminated intravascular coagulation (DIC), with estimated mortalityrates of 5% to 15%. A moderate form of HFRS caused by Seoul virus(which, along with its host, is distributed worldwide) is responsiblefor thousands of Eurasian cases annually. Serologic evidence forinfection with Seoul-like hantaviruses has been found in rodents inmajor cities of the United States, and this virus was recentlyimplicated in human cases of HFRS in Baltimore. One report has alsoassociated Seoul virus with chronic renal disease. A mild form of HFRS,caused by Puumala virus, is responsible for nephropathia epidemica inScandinavia, with an estimated mortality rate of 1% to 3%.

As used herein, a “hantavirus infection” includes any infection ordisorder associated with a hantavirus such as a Sin Nombre virus,including but not limited to hantavirus hemorrhagic fever with renalsyndrome (HFRS) (a group of clinically similar illnesses caused byspecies of hantaviruses from the family Bunyaviridae) or Hantaviruspulmonary syndrome (HPS) (an often fatal pulmonary disease which in theUnited States is caused by the Sin Nombre virus carried by deer mice).

The term “Hemorrhagic fever virus” refers to a virus which causeshemorrhagic fever from four distinct families: arenaviruses,filoviruses, bunyaviruses and flaviviruses and includes the hantavirusesas described above, ebola virus, Marburg virus, Lassa virus andCrimean-Congo hemorrhagic fever viruses.

The term “compound” is used herein to describe any specific compound orbioactive agent disclosed herein, including any and all stereoisomers(including diasteromers), individual optical isomers (enantiomers) orracemic mixtures, pharmaceutically acceptable salts and prodrug forms.The term compound herein refers to stable compounds. Within its use incontext, the term compound may refer to a single compound or a mixtureof compounds as otherwise described herein.

A “control” as used herein may be a positive or negative control asknown in the art and can refer to a control cell, tissue, sample, orsubject. The control may, for example, be examined at precisely ornearly the same time the test cell, tissue, sample, or subject isexamined. The control may also, for example, be examined at a timedistant from the time at which the test cell, tissue, sample, or subjectis examined, and the results of the examination of the control may berecorded so that the recorded results may be compared with resultsobtained by examination of a test cell, tissue, sample, or subject. Forinstance, as can be appreciated by a skilled artisan, a control maycomprise data from one or more control subjects that is stored in areference database. The control may be a subject who is similar to thetest subject (for instance, may be of the same gender, same race, samegeneral age and/or same general health) but who is known to not have afibrotic disease. As can be appreciated by a skilled artisan, themethods of the invention can also be modified to compare a test subjectto a control subject who is similar to the test subject (for instance,may be of the same gender, same race, same general age and/or samegeneral health) but who is known to express symptoms of a disease. Inthis embodiment, a diagnosis of a disease or staging of a disease can bemade by determining whether protein or gene expression levels asdescribed herein are statistically similar between the test and controlsubjects.

Purely by way of example, comparing measured levels of aninfection-associated-GTPase in a sample to corresponding control levels,or comparing measured thrombin and/or plasmin levels to control thrombinand/or plasmin levels determined in a healthy control subject, anddetermining that a subject suffers from sepsis or a hantavirus infectionor that a subject's sepsis or hantavirus infection is progressing, caninclude determinations based on comparative level differences of aboutbetween about 5-10%, or about 10-15%, or about 15-20%, or about 20-25%,or about 25-30%, or about 30-35%, or about 35-40%, or about 40-45%, orabout 45-50%, or about 50-55%, or about 55-60%, or about 60-65%, orabout 65-70%, or about 70-75%, or about 75-80%, or about 80-85%, orabout 85-90%, or about 90-95%, or about 95-100%, or about 100-110%, orabout 110-120%, or about 120-130%, or about 130-140%, or about 140-150%,or about 150-160%, or about 160-170%, or about 170-180%, or about180-190%, or 190-200%, or 200-210%, or 210-220%, or 220-230%, or230-240%, or 240-250%, or 250-260%, or about 260-270%, or about270-280%, or about 280-290%, or about 290-300%, or differences of aboutbetween about ±50% to about ±0.5%, or about ±45% to about ±1%, or about±40% to about ±1.5%, or about ±35% to about ±2.0%, or about 30% to about2.5%, or about 25% to about 3.0%, or about 20% to about 3.5%, or about15% to about 4.0%, or about 10% to about 5.0%, or about ±9% to about±1.0%, or about ±8% to about ±2%, or about ±7% to about ±3%, or about±6% to about 5%, or about 5%, or about 4.5%, or about 4.0%, or about3.5%, or about 3.0%, or about 2.5%, or about 2.0%, or about 1.5%, orabout 1.0%.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology. ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986. “Animal Cell Culture”; IRL Press, 1986. “Immobilized CellsAnd Enzymes” Perbal, 1984, “A Practical Guide To Molecular Cloning.”

A “biological sample” can be a tissue sample or a cell sample, and mostpreferably is a plasma sample.

As used herein, the terms “nucleotide” and “polynucleotide” referrespectively to monomeric or polymeric form of nucleotides of anylength, either ribonucleotides or deoxynucleotides, and include bothdouble- and single-stranded DNA and RNA. A nucleotide or polynucleotidemay include nucleotide sequences having different functions, such ascoding regions, and non-coding regions such as regulatory sequences(e.g., promoters or transcriptional terminators). A polynucleotide canbe obtained directly from a natural source, or can be prepared with theaid of recombinant, enzymatic, or chemical techniques. A nucleotide orpolynucleotide can be linear or circular in topology. A nucleotide orpolynucleotide can be, for example, a portion of a vector, such as anexpression or cloning vector, or a fragment.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers(e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, andprotein are all included within the definition of polypeptide and theseterms are used interchangeably. It should be understood that these termsdo not connote a specific length of a polymer of amino acids, nor arethey intended to imply or distinguish whether the polypeptide isproduced using recombinant techniques, chemical or enzymatic synthesis,or is naturally occurring.

A “ligand” can be any natural or synthetic moiety, including but notlimited to a small molecule, an antibody, a nucleic acid, an amino acid,a protein (e.g. an enzyme) or a hormone that binds to a cell, preferablyat a receptor (binding site) located on the surface of the cell. Theterm “ligand” therefore includes any targeting active species (compoundor moiety, e.g. antigen) which binds to a moiety (preferably a receptor)on, in or associated with a cell. In some embodiments, a ligand is apeptide, a polypeptide including an antibody or antibody fragment, anaptamer, or a carbohydrate, among other species which bind to a targetedcell.

“Binding site” as used herein is not limited to receptor protein surfaceareas that interact directly with ligands, but also includes any atomicsequence, whether or not on the surface of a receptor, that isimplicated (by affecting conformation or otherwise) in ligand binding. Apurely illustrative list of binding sites include those targeted bydetector antibodies which are specific to the GTPase-specificantibodies, and those targeted by GTPase-specific antibodies, asillustrated by the antibodies described in the Examples herein and asotherwise identifiable by techniques which are well-known to those ofordinary skill in the art.

Diagnostic methods of the present invention utilize an antibody,preferably, a monocolonal antibody, capable of specifically binding to aprotein as described herein or active fragments thereof. The method ofutilizing an antibody to measure the levels of protein allows fornon-invasive diagnosis of the pathological states of sepsis and/or ahantavirus infection. In a preferred embodiment of the presentinvention, the antibody is human or is humanized. Humanized antibodiesare antibodies, or antibody fragments, that have the same bindingspecificity as a parent antibody, (i.e., typically of mouse origin) andincreased human characteristics. Humanized antibodies may be obtained,for example, by chain shuffling or by using phage display technology.For example, a polypeptide comprising a heavy or light chain variabledomain of a non-human antibody specific for a disease related protein iscombined with a repertoire of human complementary (light or heavy) chainvariable domains. Hybrid pairings specific for the antigen of interestare selected. Human chains from the selected pairings may then becombined with a repertoire of human complementary variable domains(heavy or light) and humanized antibody polypeptide dimers can beselected for binding specificity for an antigen. Techniques describedfor generation of humanized antibodies that can be used in the method ofthe present invention are disclosed in, for example, U.S. Pat. Nos.5,565,332; 5,585,089; 5,694,761; and 5,693,762. Furthermore, techniquesdescribed for the production of human antibodies in transgenic mice aredescribed in, for example, U.S. Pat. Nos. 5,545,806 and 5,569,825.

“Fluorophores” small molecule fluors and proteinaceous fluors (e.g.green fluorescent proteins and derivatives thereof). Useful fluorophoresinclude, but are not limited to, 1,1′-diethyl-2,2′-cyanine iodide,1,2-diphenylacetylene, 1,4-diphenylbutadiene, 1,6-Diphenylhexatriene,2-Methylbenzoxazole, 2,5-Diphenyloxazole (PPO),4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM),4-Dimethylamino-4′-nitrostilbene, 4′,6-Diamidino-2-phenylindole (DAPI),5-ROX, 7-AAD, 7-Benzvlamino-4-nitrobenz-2-oxa-1,3-diazole,7-Methoxvcoumarin-4-acetic acid, 9,10-Bis(phenylethynyl)anthracene,9,10-Diphenylanthracene, Acridine Orange, Acridine yellow, Adenine,Allophycocyanin (APC), AMCA, AmCyan, Anthracene, Anthraquinone, APC,Auramine O, Azobenzene, Benzene, Benzoquinone, Beta-carotene, Bilirubin,Biphenyl, BO-PRO-1, BOBO-1, BODIPY FL, Calcium Green-1, Cascade Blue™,Cascade Yellow™, Chlorophyll a, Chlorophyll b, Chromomycin, Coumarin.Coumarin 1, Coumarin 30, Coumarin 314, Coumarin 343, Coumarin 6, Cresylviolet perchlorate, Cryptocyanine, Crystal violet, Cy2, Cy3, Cy3.5, Cy5,Cy5.5, Cy7, Cytosine, DA, Dansyl glycine, DAPI, Dil, DiO, DiOCn,Diprotonated-tetraphenylporphyrin, DsRed, EDANS, Eosin, Erythrosin.Ethidium Monoazide, Ethyl p-dimethylaminobenzoate, FAM, Ferrocene, FI,Fluo-3, Fluo-4, Fluorescein, Fluorescein isothiocyanate (FITC), Fura-2,Guanine, HcRed, Hematin, Histidine, Hoechst, Hoechst 33258, Hoechst33342, IAEDANS, Indo-1, Indocarbocyanine (C3) dye. Indodicarbocyanine(C5) dye, Indotricarbocyanine (C7) dye, LC Red 640, LC Red 705, Luciferyellow. LysoSensor Yellow/Blue, Magnesium octaethylporphyrin, Magnesiumoctaethylporphyrin (MgOEP), Magnesium phthalocyanine (MgPc), Magnesiumtetramesitylporphyrin (MgTMP), Magnesium tetraphenylporphyrin (MgTPP).Malachite green, Marina Blue®, Merocyanine 540, Methyl-coumarin,MitoTracker Red,N,N′-Difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrrin,N,N′-Difluoroboryl-1,9-dimethyl-5-[(4-(2-trimethylsilylethynyl),N,N′-Difluoroboryl-1,9-dimethyl-5-phenydipymn, Naphthalene, Nile Blue,Nile Red, Octaethylporphyrin, Oregon green, Oxacarbocyanine (C3) dye,Oxadicarbocyanine (C5) dye, Oxatricarbocyanine (C7) dye, Oxazine 1,Oxazine 170, p-Quaterphenyl, p-Terphenyl, Pacific Blue®, Peridininchlorophyll protein complex (PerCP), Perylene, Phenol, Phenylalanine.Phthalocyanine (Pc), Pinacyanol iodide, Piroxicam, POPOP, Porphin,Proflavin, Propidium iodide, Pyrene, Pyronin Y, Pyrrole, Quininesulfate, R-Phycoerythrin (PE), Rhodamine, Rhodamine 123, Rhodamine 6G,Riboflavin, Rose bengal, SNARF®, Squarylium dye III, Stains-all,Stilbene, Sulforhodamine 101, SYTOX Blue, TAMRA,Tetra-t-butylazaporphine, Tetra-t-butylnaphthalocyanine,Tetrakis(2,6-dichlorophenyl)porphyrin, Tetrakis(o-aminophenyl)porphyrin,Tetramesitylporphyrin (TMP), tetramethylrhodamine, Tetraphenylporphyrin(TPP), Texas Red®(TR), hiacarbocyanine (C3) dye, Thiadicarbocy anine(C5) dye, Thiatricarbocyanine (C7) dye, Thiazole Orange, Thymine,TO-PRO®-3, Toluene, TOTO-3, TR, Tris(2,2′-bipyridvl)ruthenium(II),TRITC, TRP, Tryptophan, Tyrosine, Uracil, Vitamin B12, YO-PRO-1, YOYO-1,Zinc octaethylporphyrin (ZnOEP), Zinc phthalocyanine (ZnPc). Zinctetramesitylporphyrin (ZnTMP), Zinc tetramesitylporphvrin radicalcation, and Zinc tetraphenylporphyrin (ZnTPP). Suitable optical dyes aredescribed in the 1996 Molecular Probes Handbook by Richard P. Haugland,hereby expressly incorporated by reference.

In some embodiments, one of the fluorescent dyes may be an Alexa Fluor®dye, including Alexa Fluor®, Alexa Fluor® 405, Alexa Fluor Alexa Fluor®430, Alexa Fluor Alexa Fluor® 488, Alexa Fluor®500, Alexa Fluor®514,Alexa Fluor®532, Alexa Fluor®546, Alexa Fluor®555, Alexa Fluor®568,Alexa Fluor®594, Alexa Fluor®610, Alexa Fluor®633, Alexa Fluor® 647,Alexa Fluor®660, Alexa Fluor®680, Alexa Fluor®700, and Alexa Fluor®750(Life Technologies Corporation Carlsbad, Calif.).

One of the fluorescent dye may be a tandem fluorophore conjugate,including Cy5-PE, Cy5.5-PE, Cy7-PE, Cy5.5-APC, Cy7-APC, Cy5.5-PerCP,Alexa Fluor®610-PE, Alexa Fluor® 700-APC, and Texas Red-PE. Tandemconjugates are less stable than monomeric fluorophores, so comparing adetection reagent labeled with a tandem conjugate to reference solutionsmay yield MESF calibration constants with less precision than if amonomeric fluorophore had been used.

The flurophores may be a fluorescent protein such as green fluorescentprotein (GFP; Chalfie, et al., Science 263(5148):802-805 (Feb. 11,1994); and EGFP; Clontech—Genbank Accession Number U55762), bluefluorescent protein (BFP; Quantum Biotechnologies, Inc. Montreal Canada;Stauber, R. H. Biotechniques 24(3):462-471 (1998); Heim, R. and Tsien,R. Y. Curr. Biol. 6:178-182 (1996)), cyan fluorescent protein (CFP) andenhanced yellow fluorescent protein (EYFP; Clontech Laboratories, Inc.,Palo Alto, Calif.). In some embodiments, the fluorescent dye is dTomato,FlAsH, mBanana, mCherry, mHoneydew, mOrange, mPlum, mStrawberry,mTangerine, ReAsH, Sapphire, mKO, mCitrine, Cerulean, Ypet, tdTomato,Emerald. or T-Sapphire (Shaner et al., Nature Methods, 2(12):905-9.(2005)).

The flurophores may be a fluorescent dye in the form of a fluorescentsemiconductor nanocrystal particle, or quantum dot, including Qdot®525nanocrystals, Qdot®565 nanocrystals, Qdot®585 nanocrystals, Qdot®605nanocrystals, Qdot®655 nanocrystals, Qdot®705 nanocrystals, Qdot®800nanocrystals (Life Technologies Corporation, Carlsbad, Calif.). In someembodiments, the fluorescent dye may be an upconversion nanocrystal, asdescribed in Wang et al., Chem. Soc. Rev., 38:976-989 (2009).

The fluorescent molecules (fluorophores) may be conjugated withantibodies or other detection reagents, and associated with componentsof a sample that is analyzed by the instrument. Fluorophores can beactivated by light from the instrument and re-emit light of a differentwavelength. Since antibodies bind to antigens on the cells, the amountof light detected from the fluorophores is related to the number ofantigens associated with the cell passing through the beam. In anotherembodiment of the invention, a fluorescently-labeled DNA oligonucleotidecan be associated with the genomic DNA of a cell, and the amount oflight detected from the fluorophores is related to the number of copiesof the oligonucleotide that have hybridized to complimentary regions inthe genome. Any specific set of fluorescently tagged detection reagentsin any embodiment can depend on the types of experimental samples to bestudied. See United States Patent Application Document No. 20130109050.As further explained in United States Patent Application Document No.20130109050, “[s]everal fluorescent detection reagents can be usedsimultaneously, so measurements made as one cell passes through thelaser beam consist of scattered light intensities as well as lightintensities from each of the fluorophores. Thus, the characterization ofa single cell can consist of a set of measured light intensities thatmay be represented as a coordinate position in a multidimensional space.Considering only the light from the fluorophores, there is onecoordinate axis corresponding to each of the fluorescently taggeddetection reagents. The number of coordinate axes (the dimension of thespace) is the number of fluorophores used. Modem flow cytometers canmeasure several colors associated with different fluorophores andthousands of cells per second. Thus, the data from one subject can bedescribed by a collection of measurements related to the number ofantigens for each of (typically) many thousands of individual cells. SeeU.S. Pat. Nos. 7,381,535 and 7,393,656 for examples of flow cytometrymethods and applications, which are hereby incorporated by reference intheir entirety.”

Those of ordinary skill in the art know how to select a secondfluorophore that has a wavelength (color) which is different from thatof the first fluorophore as required in various embodiments of themethods described and claimed herein.

The Rho family of GTPases belongs to the Ras superfamily of lowmolecular weight (˜21 kDa) guanine nucleotide binding proteins. The mostextensively studied members are RhoA, Rac1, and Cdc42. Small GTPasesarea family of hydrolase enzymes that can bind and hydrolyze guanosinetriphosphate (GTP). Small GTPases are a type of G-protein found in thecytosol; a small GTPase can function independently as a hydrolase enzymeto bind to and hydrolyze a guanosine triphosphate (GTP) to formguanosine diphosphate (GDP).

The Rho-family of p21 small GTPases are directly linked to theregulation of actin-based motile machinery and play a key role in thecontrol of cell migration. Aside from the original and mostwell-characterized canonical Rho GTPases RhoA, Rac1, and Cdc42, numerousisoforms of these key proteins have been identified and shown to havespecific roles in regulating various cellular motility processes.

A typical G-protein is active when bound to GTP and inactive when boundto GDP (i.e. when the GTP is hydrolyzed to GDP). The GDP can be thenreplaced by free GTP. Therefore, a G-protein can be switched on and off.GTP hydrolysis is accelerated by GTPase activating proteins (GAPs),while GTP exchange is catalyzed by Guanine nucleotide exchange factors(GEFs). Activation of a GEF typically activates its cognate G-protein,while activation of a GAP results in inactivation of the cognateG-protein. Guanosine nucleotide dissociation inhibitors (GDI) maintainsmall GTP-ases in the inactive state.

The GTPase RhoA effectors include:

Cit, Cnksr1, Diaph1, Diaph2, DgkQ, FInA, KcnA2, Ktn1, Rtkn1, Rtkn2,Rhpn1, Rhpn2, Itprl1, PlcG1, PI-5-p5K, Pld1, Pkn1, Pkn2, Rock1, Rock2,PrkcA and Ppp1r12A.

The GTPase Rac1 effectors include Sra1, IRSp53, PAK1, PAK2 and PAK3.

The GTPase Cdc42 effectors include Wiskott-Aldrich syndrome protein.N-WASP, IRSp53, Dia2, Dia3, ROCK1 and ROCK2.

In a preferred embodiment, effector proteins are selected from the groupconsisting of PAK-1 RBD (a Rac1 and Cdc42 effector), Raf-1 RBD, (a Raseffector). Rhotekin-RBD (a Rho effector), RalGDS-RBD (a RAP1 effectorprotein) and RILP-RBD (a Rab-7 effector protein).

The present invention therefore provides rapid, effector-based,flow-cytometry assays to quickly assess sepsis-related GTPase activationstatus and monitor multiple GTPases in a single sample. In particular,the present invention relates to a novel flow cytometry-compatible,bead-based effector-binding assay for rapidly monitoring the activationstatus of multiple Ras superfamily GTPases in cell lysates. We havedemonstrated proof-of-principle through the use of known agonists andantagonists of individual GTPases.

Additionally, we have applied the assay to acquire new information aboutthe temporal activation of multiple GTPase cascades following SNVexposure and internalization that offer previously unprecedentedmechanistic detail about SNV induced activation of cellular signaling.The data show that the interaction of hantaviruses with cognate, cellsurface localized receptors responsible for entry results in theactivation of signaling cascades that converge in the sequentialactivation of multiple small GTPases in the Ras, Rho and Rab families.In this way the GTPases act as signaling hubs²⁹ that further propagatethe signals first induced by SNV cell surface receptor binding topromote the requisite changes in cell-cell adhesion, integrin activationand endocytosis that are necessary for virus infection and replication.

The invention is illustrated further in the following non-limitingexamples.

Example I

Effector Flow Cytometry Bead (EFCB) Assay

A novel flow based assay was platformed by monitoring GTPase activationin two different cell types (Vero E6 cells as well as HeLa cells usedfor-Rab7 experiments), suggesting that the assay is readilygeneralizable to different cell types. By coating beads with differenteffectors and multiplex analysis of fluorescent bead sets bearingdifferent effectors³⁰ the assay can be extended to the simultaneous andhigh throughput analysis of multiple GTPases from the same cell lysatesample. We therefore anticipate that this approach will enableinvestigations into the interconnection of signaling networks withGTPase activation in normal cellular functions as compared to viral orbacterial infections or other pathogenic processes.

For productive infection of host cells, viruses typically activatemultiple cellular GTPases to promote the cytoskeletal remodelingrequired for breaching inter- and intracellular cellular barriers toinfection as well as intracellular trafficking of internalized virionsto allow replication. We are particularly interested in Hantavirusesthat cause hemorrhagic fever with renal syndrome (HFRS) and hantaviruscardiopulmonary syndrome (HCPS).¹⁻⁴ While the mechanism of hantavirusentry into host cells and the subsequent pathogenesis are poorlyunderstood, a_(v)b3 integrin and attachment factor, decay-acceleratingfactor (DAF/CD55) are known mediators of cell entry. DAF is aglycosylphosphatidylinostol (GPI)-anchored surface protein that protectscells from lysis by autologous complement and is highly expressed inmany cells types.⁵ When localized in lipid rafts,⁶ DAF can formcomplexes with Src family tyrosine kinases⁷⁻¹⁰ that signal upstream ofthe Ras superfamily of small GTPases which regulate many cell functions,such as cell proliferation, cytoskeletal alterations, trafficking,differentiation, survival, and migration. The ability to form closeassociation with these signaling proteins capable of altering cellbarrier function has made DAF an important receptor for pathogens. ThusDAF ligation on the cell surface might induce a signaling cascadeinvolving a coordinate network of small GTPases.¹¹ ¹² Parallelmeasurements of active GTPases downstream of receptor engagement willtherefore be important for understanding how these signals are regulatedand when and how their consolidation is related to both physiologicaland pathological contexts.

As described herein, we have provided a new flow cytometry approach forquantitatively measuring the activation status of Ras superfamilyGTPases in activated cells based on specific effector binding. Theadvantages of the effector flow cytometry bead (EFCB) assay overconventional western blot and ELISA based methods (commerciallyavailable through Cytoskeleton. Inc.) are: 1) EFCB enables rapidmeasurement and analysis with results within 4 h, compared to days; 2)it offers quantitation and increased sensitivity measuring activatedGTPases in cell lysates from <250,000 cells grown in a 48 well plate,well below the requisite minimum of 2×10⁶ cells for ELISA, and 3) it canmeasure multiple GTPases from a single lysate either sequentially orusing multiplex approach with differentially labeled beads, which cannot be achieved with conventional assays that require the entire lysatefor measurement of a single GTPase assay. For EFCB we functionalizedglutathione beads with GST tagged Ras binding domains (RBD) of theeffectors for Rac1 & Cdc42, H-Ras & R-Ras, Rap1, RhoA and Rab7: namely.PAK1, Raf1, Ral, RhoTekin, and RILP respectively. In this way uniqueeffector-bearing beads were used as bait to extract active GTPasesrequired for cytoskeletal remodelling (Rac1 and RhoA), regulation ofadhesion (Rap1 and Ras) and trafficking (Rab1) from the same cell-lysatesample. The bead-bound effector-GTPase complexes were then incubatedwith fluorescently labeled antibodies directed against each activatedGTPase and analyzed on a flow cytometer.

Exposing Vero E6 cells to known titers of UV killed and fluorescentlylabeled Sin Nombre hantavirus particles caused cells to first formfilopodia and lamellipodia then contract and lose adhesion (FIG. 1a-d.).¹³ These morphological changes are associated with Cdc42 (filopodia)Rac1 (lamellipodia), and RhoA activity manifested as stress fiberformation and cellular contraction. Using Pak1 and Rhotekin beads wereused to record ≥2-fold increases in active Rac 1 and RhoA above restinglevels in virus treated cells at 3 min and then returned to basal levelsat twenty minutes. Similar measurements were recapitulated in celllysates treated with known activation and inhibition standards of theGTPases. For brevity and clarity we will focus RhoA treatment. Calpeptinand EGF where used to activate RhoA while RhoGAP was used to hydrolyzeactive RhoA. In addition, owing to the known mutual antagonism betweenRhoA and Rac1, NSC23766 a specific inhibitor of the Rac1 GEF, Tiam1, wasused to assess the effect of Rac1 suppression on the expression ofactive RhoA in EGF activated cells. Using the EFCB assay, a >2 foldincrease in active RhoA was measured in calpeptin treated cells. ActiveRhoA was hydrolyzed to baseline levels with RhoGAP. In EGF treatedcells, NSC23766 suppressed active Rac1 to a degree below the GTPase'sbasal activity. From the same lysates, parallel measurements of RhoA-GTPshowed a 2-fold increase above basal levels and 1.3 above EGFstimulation alone. The data are consistent with the mutual antagonism ofRac1 and RhoA wherein the inhibition of Rac1 is sensed and results in ahigher RhoA activation.¹⁴

We also used a plate based GLISA assay to measure Rho family GTPaseactivation as a function of virus stimulation in normal cells and cellswhere surface-expressed DAF was first cleaved through PI-PLCtreatment.¹⁵ Virus exposure resulted in a significant time-dependentactivation of RhoA and Rac1 GTPases peaking between 7 and 15 min afterexposure (FIG. 4A). Rac1 GTPase activation was abolished in DAF-clearedcells demonstrating that the engagement of a GPI anchored receptor suchas DAF was required upstream of Rac1. The overlapping temporal responsein Rac1 and RhoA activation is consistent with cellular responseelicited by Group B Coxsackieviruses, which are also ligands of DAF.⁶ Atlonger times after virus exposure, the levels of active, GTP-bound Rac1and RhoA fell below the baseline levels of active GTPases in uninfectedcontrols. The diminution of GTPase activity, might be due to loss ofcell adhesion, due to integrin anchorage regulatory role of Rac1-GTP.¹⁶¹⁷ Collectively, the controls and the GLISA assay data demonstrateapplicability of our EFCB assay the measurement of active GTPases.

The loss of cell-cell adhesion in virus-activated cells led us toexamine the relationship between loss of adhesion and changes inintegrin affinity states across the ventral surface of virus activatedcells. With this in mind we used our EFCB assay to examine the activityof Rap1 and H-Ras, which are known to play antagonistic roles inintegrin affinity regulation.¹⁸⁻²² Glutathione beads functionalized witheffectors for Rap1 (GST Ral-GDS) and and H-Ras (Raf-1 RBD) were used tosimultaneously assay activated Rap1, and H-Ras GTPases from virusactivated cell lysates. Cocurrently, we used FTI277 a specific inhibitorof H-Ras and 8-Cpt-2m-cAMP, and activator of Rap1 as a validationcontrols, which also exhibit antagonism between H-Ras and Rap1 with Rap1interfering with the Raf effector function of H-Ras. Thus inhibition ofH-Ras induced a near 2-fold increase active Rap1, while activation ofRap1 decreased basal levels of H-Ras. Consistent with theseobservations, the activity of Rap and H-Ras in virus-activated celllysates at 3 min and 20 min post-exposure showed that the pair wasactivated sequentially, where active Rap 1 peaked within the first 3 minand subsequently deactivated at 20 mins while H-Ras reamined low whileRap1 was at its zenith, eventually peaking at 20 min as Rap1 was indecline. The staggered activity of Rap1 and H-Ras appears to follow themorphological trend established for the RhoGTPase assay in FIG. 1, wherethe loss of cell adhesion observed towards the end of the measurement isconsistant with H-Ras upregulation, which is upstream of the downregulation of integrin affinity.

We next tested the applicability of the EFCB assay to monitor theintracellular trafficking of viruses by examining the activation of Rab7in cells exposed to SNV. Rab7 and its downstream effector, Rab7interacting lysosomal protein (RILP) are involved in the regulation ofendocytic transport at several stages, post-intemalization.^(24, 25)First, EGF was used to demonstrate the tractability of monitoringcellular Rab7 activation using beads functionalized with RILP-RBD (FIG.3a ). Within 15 min following EGF stimulation in HeLa cells, Rab7activity was nearly 10 fold higher than in resting cells, consistentwith the time course required for EGF-EGFR complex to reach lateendosomes.²⁶ Next the Rab7-GTP levels were assessed in Vero E6 celllysates following exposure to virus particles for various time intervalsbetween 0 and 60 min. The highest Rab7-GTP levels were realized at 20min after virus exposure (FIG. 3b ). The timing of Rab7 activity isconsistent with the endocytic delivery of large viral cargo toRab7-positive late endosomes, which occurs in the 10-20 min timeframe.^(27, 28) Immunofluorescence images of Rab7-positive endosomesafter the synchronized entry of fluorescently labeled SNV (FIG. 5),shows that endosomes are redistributed from perinuclear space to thecell periphery and colocalize with cargo in the same time range.

In sum, we have devised a novel flow cytometry-compatible, bead-basedeffector-binding assay for rapidly monitoring the activation status ofmultiple Ras superfamily GTPases in cell lysates. We have demonstratedproof-of-principle through the use of known agonists and antagonists ofindividual GTPases. Additionally, we have applied the assay to acquirenew information about the temporal activation of multiple GTPasecascades following SNV exposure and internalization that offerpreviously unprecedented mechanistic detail about SNV induced activationof cellular signaling. The data show that the interaction ofhantaviruses with cognate, cell surface localized receptors responsiblefor entry results in the activation of signaling cascades that convergein the sequential activation of multiple small GTPases in the Ras, Rhoand Rab families. In this way the GTPases act as signaling hubs²⁹ thatfurther propagate the signals first induced by SNV cell surface receptorbinding to promote the requisite changes in cell-cell adhesion, integrinactivation and endocytosis that are necessary for virus infection andreplication.

Our novel flow based assay was platformed here by monitoring GTPaseactivation in two different cell types (Vero E6 cells as well as HeLacells used for-Rab7 experiments), suggesting that the assay is readilygeneralizable to different cell types. By coating beads with differenteffectors and multiplex analysis of fluorescent bead sets bearingdifferent effectors³⁰ the assay can be extended to the simultaneous andhigh throughput analysis of multiple GTPases from the same cell lysatesample. We therefore anticipate that this approach will enableinvestigations into the interconnection of signaling networks withGTPase activation in normal cellular functions as compared to viral orbacterial infections or other pathogenic processes.

Materials and Methods

Antibodies. Monoclonal rabbit anti-RAP1 was obtained from Santa CruzBiotechnology, Inc (Santa Cruz, Calif.). Monoclonal mouse antibodies:anti-Rho (A,B,C) clone 55, anti-Rac1, anti-Rab7 including the secondaryantibody goat anti-mouse IgG (H+L) conjugated to Alexa Fluor 488 werewere obtained from Millipore (Temecula, Calif.). Monoclonal mouseAnti-RRAS antibody was obtained from Abcam (Cambridge, Mass.). AntiRab 7antibody (Sigma) Activators and Inhibitors. Rap Activator 8-Cpt-2me-cAMP(50 μM) obtained from R & D Systems (Minneapolis, Minn.). RacInhibitor(OOpM NSC23766 from Calbiochem). FTI-277 trifluoroacetate salt(an inhibitor of Famesvl Transferase of H-Ras and K-Ras; 50 nM), andRap1 inhibitor GGTI 298 trifluoroacetate salt hydrate, was obtained fromSigma-Aldrich (St. Louis, Mo.). Calpeptin (0.3 μM Rho activator) wasfrom Calbiochem, Inc. 20 μg RhoGAP from Cytskeleton and Recombinant EGFwas from Invitrogen.

Buffers: RIPA buffer: 74 mM CaCl₂, 50 mM Tris-HCl pH 7.4, 1% NP-40, 0.5%deoxycholic acid, 0.1% SDS, 1 mM sodium orthovanadate, and proteaseinhibitors. HHB buffer: 7.98 g/L HEPES (Na salt), 6.43 g/L NaCl, 0.75g/L KCl, 0.095 g/L MgCl₂ and 1.802 g/L glucose. Cell Culture: HeLa andVero E6 cells from ATCC are plated at 4×10⁵ cells per T75 flask, thenallowed to grow for ˜48 hours to medium/low confluence in Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10% FBS, pen/strep, andL-glutamine in a 37 degree C. incubator with 5% CO₂. Cells wereserum-starved in DMEM overnight before the GST pull-down.

Production of Sin Nombre Virus. SNV was propagated and titered in VeroE6 cells under strict standard operating procedures using biosafetylevel 3 (BSL3) facilities and practices (CDC registration numberC20041018-0267) as previously described³¹. For preparation ofUV-inactivated SNV, we placed 100 μL of virus stock (typically 1.5-2×10⁶focus forming units/ml) in each well of a %-well plate and subjected thevirus to UV irradiation at 254 nm for various time intervals (˜5 mW/cm²)as described elsewhere³². We verified efficiency of virus inactivationby focus assay before removing from the BSL-3 facility.

Fluorescent Labeling of SNV. The envelope membrane of hantavirusparticles was stained with the lipophilic lipid probe octadecylrhodamine(R18) and purified as previously described³². The typical yield of viralpreparation was 1±0.5×0 particles/μl in 300 μl tagged with ˜10,000 R18probes/particle or 2.7 mole % R18 probes in the envelope membrane ofeach particle of 192 nm diameter average size. Samples were aliquotedand stored in 0.1% HSA HHB buffer, and used within two days ofpreparation and storage at 4° C. For long-term storage, small aliquotssuitable for single use were stored at −80° C.

Effector Proteins. GST-effector chimeras used for the studies were asfollows. PAK-1 PBD, a Rac1 effector and Raf-1 RBD, a RAS effectorprotein were obtained from Milipore (Temecula, Calif.). Rhotekin-RBD, aRho effector protein was purchased from Cytoskeleton, Inc. (Denver,Colo.). Ral-GDS, a RAP1 effector protein was obtained from ThermoScientific (Fair Lawn, N.J.). RhoGEF was obtained from Santa CruzBiotechnology, Inc. (Santa Cruz, Calif.).

Expression and purification of GST-RILP. Plasmid encoding GST-RILP wasexpressed in competent E. coli BL21 cells. Cultures were grown at 37° C.to an absorbance of 0.5 O.D, measured at 595 nm and protein expressioninduced by transfer to room temperature and addition of 0.2 mMisopropyl-beta-D-1-thiogalactopyranoside (IPTG) for 16-18 h to maximizeyield of properly folded active fusion protein. Purification of GST-RILPwere performed according to standard procedures as previouslydescribed.³³ ³⁴ The harvested bacterial cell pellet was resuspended incold native binding buffer. Cells were lysed using lysozyme and microtipsonicator (Misonix Inc., Newtown, Conn., U.S.A.). The cell lysates weremixed with 10% TritonX-00 and mix over end for 30 min at 4° C. Cellswere then centrifuged at 8,000×g for 10 minutes to pellet the cellulardebris. The supernatant was mixed with freshly prepared GlutathioneSpharose 4B slurry and bound at 4° C. for 2 h using gentle agitation tokeep the resin suspended in the lysate solution. Glutathione beads withbound protein was settled using low speed centrifugation (500×g)followed by multiple washes before eluting the glutathione sepharosebound GST-tagged protein with 10 mM Glutathione in 50 mM Tris-HClelution buffer. Eluted protein was concentrated by passing throughMillipore Amplicon Ultra Tubes (MWCO 30,000). The purified protein wasquantified using the BCA protein estimation kit (Pierce). Single usealiquots stored at −80° C. were used in the experiments.

Assembly of GST Effector proteins on GSH beads and GST pull-down assay.13 μm Superdex peptide beads were derivatized with glutathione(γ-glu-cys-gly or GSH) as previously described.³³ GST conjugates ofvarious GTPase-effector proteins were bound to suspensions of GSH beadsto in order to form molecular assemblies necessary for capturingGTP-bound GTPases from cell lysates. The characteristic kinetic andequilibrium binding constants of glutathione-S-transferase (GST) fusedto Green fluorescent protein (GFP), were used to establish optimalstoichiometric mixtures of GSH beads and specific GST effector fusionproteins for desired site occupancies of the GST effector proteins usedin the flow cytometry assays.³³ Briefly, the binding assay of GST GFP toGSH beads was used to establish the following parameters. The saturablesite occupancy values were used to determine the concentration ofbead-borne GSH, while the K_(d)˜80 nM was used to establish the optimalconcentration (10×K_(d)) of GST effector proteins required to saturateGSH sites on the beads. GSH beads were mixed with the desired effectorprotein, incubated with shaking for 2 h at 4° C., recollected bycentrifugation, and resuspended in RIPA buffer to 10,000 beads/μL. Beadswere prepared fresh for each experiment and kept on ice while celllysates were prepared.

TABLE 1 SNV Assay Results Activator; final Inhibitor; finalconcentration; concentration; SNV titer; GTPase Effector time time timeRhoA Rhotekin- Calpeptin; 1 μM; 10,000/cell; RBD, 30 min 3, 10, 20, 30,60 Rac1 PAK-1 RBD EGF; 10 nM; NSC23766, 15 min 100 μM, 30 min Rap1Ral-GDS 8-Cpt-2me- GGTI 298 cAMP; 50 μM; 30 min R-Ras Raf-1 RBD H-RasRaf-1 RBD FTI-277; 100 nM; 30 min Rab-7 RILP-RBD EGF; 10 nM; 15 min

Stimulation and cell lysis. Vero E6 or HeLa cells were plated in 48 wellplates or T 75 flasks and incubated overnight at 37° C. with 5% CO₂ and90% humidity. Cells were starved for overnight in serum free mediumfollowed by stimulation with activator or inhibitor at concentrationsand times shown in Table 1. Stimulation with virus was typicallyperformed with titers 10,000 particles/cell co-administered with aninhibitor as necessary and incubated for times ranging from 3 to 60 minas necessary. After incubating cells with various inhibitors,activators, and/or SNV^(R18) for desired times, cells were lysed with300 μL ice-cold RIPA buffer. The flasks are scraped, and the lysatecollected. For the purposes of standardization, a fraction of thesupernatant was collected, quantified using BCA protein estimation kit(Pierce). Lysates were kept ice cold at all times to limit hydrolysis ofactive GTPases. In our hands Rap1 was especially susceptible tohydrolysis whereas most of the other GTPases were robust. Lysates weresonicated briefly, and then centrifuged at 14.000 rpm for 10 minutes toclear the lysate of any unlysed materials and DNA. 10,000 beads for eacheffector assay was used and added to a lysate. If probing for more thanone protein, divide the lysate was devided into desired tubes and probeindividually. The beads and lysate were allowed to incubate for 1 hourat 4° C. After incubation, the beads were pelleted in a cold centrifuge.The residual lysate was collected and re-assayed for a different GTPaseusing different effector beads. The lysates could be saved for furtherpull down reactions by flash freezing in liquid nitrogen, then storingat −20 degrees C. for no longer than a week. The bead pellets wereresuspended in RIPA buffer and incubated with a primary monoclonalantibody for the target GTPase, with gentle shaking for 1 hour. Thebeads were than centrifuged and Pellet resuspended in HHB buffer with anAlexa 488 secondary antibody at a 1:200 dilution for 1 hour with shakingat 4 degrees. Finally the beads were centrifuged once and resuspended in100 uL RIPA buffer for reading on the flow cytometer.

Confocal Microscopy. Confocal laser scanning microscopy was performedwith Zeiss META or LSM 510 systems using 63×1.4 oil immersion objectivesas previously described³⁵. For live cell imaging, Vero E6 cells wereplated in 8-well Lab-Tek chambers (Nunc) and temperature was maintainedat 30° C. with an objective heater (Bioscience Tools) in appropriatebuffer containing the desired cations, Ca²⁺ or Mn²⁺, which respectivelyconfer low and high affinity states of a_(v)b₃ ³⁵. In situ addition ofSNV^(R18) was performed by micropipeting an aliquot (100 μL) of ≈1×10⁹virion particles per well containing ≈1.5×10⁵ cells (this is nominallythe stoichiometric equivalent of a multiplicity of infection (m.o.i.) of1 if the virions from our stocks had not been inactivated with UV light)in 200 μl in HBSS and mixing well by trituration with the pipet. Imageswere collected using 2× line averaging at 3-10 s intervals, depending onimaging time, where longer intervals were necessary to minimizephotobleaching.

REFERENCES

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Example 2

Flow Cytometry Assays for the Diagnosis of Sepsis.

Patient Cohort. The UNM Hospital (UNMH) is the only level 1 traumacenter in the state of NM. According to the UNMH trauma registry thepatients admitted between Jan. 1, 2008 and Dec. 31, 2012 comprised of13,144 adults. Thus, an average of 500-600 trauma patients are admittedto our ICU annually, with an average of 200-250 complicated traumapatients defined as having a length of stay greater than 5 days with ahigh injury severity score (ISS)>20. Of the complicated trauma ICUpatients, ˜100-125 patients per year had sepsis spanning the range:sepsis (Systemic Inflammatory Response (SIRS) with infection), severesepsis (organ dysfunction, or septic shock (severe sepsis withhypotension). The nature of injuries and associated infection are listedin Table 2 below.

TABLE 2 Association of pathogen with traumatic site of injury and timefrom presentation Site of injury Pathogen t_(dx) Soft tissue: stab,Staphylococcal and Streptococcal first gunshot open infection includingMRSA 5-7 days fractures etc. Gram-neg. (Enterobacter, E. coli, >7 dayslater infection on Pseudomonas, Acinetobacter) same wound Deep muscleinjury anaerobes including Bacteroides, >5 days wounds Clostridium, andPeptostreptococcus Lung based infections. gram-pos. organisms (Strep,Staph, <5 days Early: H. flu) Late: gram-neg. including Pseudomonas, >5days Acintobacter, and Serratia Intraabdominal gram neg. rod (E. coli,Enterobacter), >5 days infection (abscess gram positivie (Enterococcus),and peritonitis) gram positive rods (Clostridium, Bacteroides).

With IRB approval, patients were enrolled with the goal of collectingdaily blood samples from subjects admitted to UNMH intensive care unit(ICU) in order to investigate host responses to trauma and sepsis. Thede-identified patient records contain clinical, and demographic data(gender and ethnicity etc.) Multiple small volume aliquots of codedserial samples, collected for up to 14 days from enrollment. 25 are nowstored in a −80° C. freezer, in the PI's lab. We have also enrolled 20healthy controls with a target of 50 subjects for our study.

Inclusion criteria. Patients admitted under the Trauma Alert Protocol(TAP) to the Emergency Department (ED) at the University of New MexicoHospital will be screened for inclusion in the study. The TAP system isactivated in response to a specific constellation of traumatic injuries,which are known to predict the need for Trauma Surgical consultation.These include: a) High speed mechanism motor vehicle collisions/crashes.b) Falls from great heights. c) Penetrating trauma (stab wounds andgunshot wounds) to the proximal extremities, head, or torso, d)Significant crush injuries, e) Patients hit by motor vehicle. f) Highspeed bicycle and motorcycle crashes. g) Any other significant trauma asjudged by ambulance report.

Initial inclusion criteria include age greater than 18, TAP statusduring trauma resuscitation, not pregnant, and not currently a prisoner.Patients meeting initial inclusion criteria will have a single bloodsample drawn at the time of ED evaluation. They will then undergoscreening for final inclusion in the study within 24 hours of arrival.Secondary, or final inclusion criteria include meeting all requirementsof initial inclusion, being admitted to an intensive care unit (ICU),and possessing the ability to provide English or Spanish writteninformed consent either personally or via a surrogate decision maker asdetermined by the New Mexico Uniform Health Care Decisions Act (NMSA1978 24-7A-5).

Thrombin and Plasmin Contribute to Loss of Cell Barrier Function in CellMonolayers as Measured with Electric Cell-Substrate Impedance Sensing(ECIS).

We have recently used an electric cell-substrate impedance sensing(ECIS) based assay,^(35A) to measure the differential contributions ofthrombin and plasmin to the loss of cell barrier function in patientsinfected with Sin Nombre virus.^(35A) For ECIS measurements, Vero E6cells developed as a more robust surrogate for endothelial cells inprimary screens were plated at confluence in electrode-containing dishes(Applied BioPhysics, Inc., Troy, N.Y.) and then allowed to attach,spread, and organize for at least 24 hours. Cellular impedance wasmeasured continuously at a single frequency of 4,000 Hz. Increase inresistance corresponds to increasing cell barrier function. Whencellular impedance reached plateau values 3,000Ω-4,000Ω, 20 μl of plasmawere added to 200 μl of media in the well. FIG. 6 shows that exposure ofcells to trauma patient plasma caused cell monolayer resistance to droprapidly and then partially recover to a lower plateau over the course ofseveral hours. In contrast, plasma from healthy controls had no effect.The multiphasic changes in the time-course experiments are caused byseveral edemagenic factors such as thrombin, plasmin and otherinflammatory mediators. Pretreatment of plasma samples with selectiveinhibitors of thrombin and plasmin can be used as a surrogate forassessing hemostasis balance in trauma patients (FIG. 7).

Plasmin Activity is Unregulated in Some Patients Who Develop Sepsis.

The edemagenic signaling of thrombin and plasmin is mediated byprotease-activated receptors (PAR-1, PAR-3 and PAR-4).^(27-31A) PARreceptor-signaling causes cytoskeletal remodeling characterized by theactivation of the small GTPase, RhoA associated with stress fibers andloss of cell barrier function.^(30, 40-45A) We tested the idea that hostresponse to bacterial infection elicits an increase in thrombogenesis,(release of thrombin in circulation) which generates increasedfibrinolysis (increase in plasmin in serial samples). We used thefollowing subjects.

Subject P13: White female, 77, was admitted after a fall down stairssustaining a head injury and rib fractures, intubated on arrival in theemergency department. Clinical Summary: day 2: respiratory distress,possible pneumonia; placed on ventilator; fever; sputum culturecollected, and a broad-spectrum antibiotic treatment was initiated onday 4, day 6: MRSA infection confirmed; antibiotic treatment continuedfor 8 consecutive days. Day 8: Developed an unexplained leukocytosis to˜17000 WBC which resolved on its own. 13 days in ICU dis-enrolled fromstudy after 10 days of sample collection due to family request.

Subject P19: Hispanic white male, 52, was admitted after shotgun woundto abdomen; low grade liver lacerations, high grade right renal injury;traumatic abdominal wall hernia ˜3 cm, fracture to three ribs, 7 days inICU, no frank sepsis during 14 day sample collection.

Serial samples of patients P13 and P19 were each treated with a mixtureof thrombin and plasmin inhibitors, (argatroban^(29A) and tranexamicacid^(46A) respectively) and analyzed by ECIS (FIG. 7). Using the datapoints taken at 4 hours in the time-course assays (FIG. 7), we thencalculated the ratio of the thrombin-inhibited samples and thrombin- andplasmin-inhibited assays (Arg/Arg+TA in FIG. 8) in order to determinethe relative expression of thrombin and plasmin in the two patients.

For P19, argatroban alone produces nearly the same result as a mixtureof argatroban and tranexamic acid, suggesting that the level ofcirculating Plasmin in P19 was negligible. For P13, the recovery of cellbarrier function was better when the mixture of thrombin and plasmininhibitors was used, compared to thrombin alone (P13 in FIG. 8). Thedownward slope of the ratiometric plot suggests that plasmin increaseddaily up to day 8 when the patient presented with leukocytosis.

GTPAse Activity Screen.

We have developed a multiplex Gtrap assay^(36A) that allows us tomeasure the expression of multiple GTPases that are activated byexposure of cells to patient plasma. For proof of principle we foundthat P13 samples induced activation of RhoA and Rap 1 whereas P19 wasrelatively silent. FIG. 8B shows that the activity of RhoA measured inVero E6 cells, after exposure to plasma (P13 and P19) collected on evendays from day 0 to 8. For P13 RhoA activity peaks at day 4 and dropsthereafter. We hypothesize that the increase in thrombin generation inP13 was in response to bacterial infection as the decrease of RhoAactivity coincided with antibiotic treatment on day 4. The changes inRap1 mirrored RhoA (not shown) However, treatment of samples withargatroban shows that RhoA GTP activation was primarily due tothrombin^(27-31A) rather than direct bacterial activation.^(14A, 20A)

Onset of Sepsis in Patients with Measurement of Cell Barrier Function InVitro.

Rationale and Hypothesis. The clinical presentation of sepsis is oftenindistinguishable from systematic inflammatory response caused bysterile inflammation in trauma.^(9A) This ambiguity may cause delays inthe administration of lifesaving standard therapies of appropriateantibiotics and precludes misuse of antimicrobial agents.^(9A) Duringinfection, inflammatory mediators of bacteria and/or host can manipulatethe procoagulant/anticoagulant equilibrium and cause sepsis as well asengaging the RhoGTPases that regulate the actin cytoskeleton. We haveobserved that the onset of infection can be manifested by an increase inplasmin as measured by changes in cell monolayer resistance FIG. 2A andincrease in RhoA and Rap1 activity (FIG. 8).

Screening Serial Plasma Samples for Preseptic Changes of Cell BarrierFunction.

Experimental Approach. A 96 well plate format ECIS (Applied Biophysics,Troy, N.Y.) will be used to screen a collection of over 30-50 differentserial plasma samples being collected from patients who go on to developclinically documented cases of sepsis or sterile systematic inflammatoryresponse (SIRS). For ECIS measurement, Vero E6 cells will be plated atconfluence for at least 24 hours in electrode-containing dishes (AppliedBioPhysics, Inc., Troy, N.Y.). In our experience,^(35A) Vero E6epithelial cells derived from monkey kidney cells form more robustcell-cell barrier contacts (with typical resistance values in the3,000-4,000 ohm range) compared to endothelial cells (1,000-1.500 ohms).Compared to endothelial cells,^(35A) Vero cells are less sensitive tospurious environmental cues that disrupt cell barrier function. Whileprevious studies have shown that thrombin differentially regulates humanlung epithelial and endothelial cells^(47A) our results herein suggestthat the effects of thrombin on Vero E6 cells is similar to endothelialcells.^(35A) They grow to confluence very rapidly compared toendothelial cells and very suitable for high throughput primary screens.Experiments will be performed in triplicate wells during the same run,at any given time. The readout for these experiments will be ameasurement of changes in cell monolayer resistance after exposure toplasma samples treated with a mixture of argatroban and tranexamic acidand argatroban alone to block the activation of PAR receptors bythrombin and plasmin as described for FIG. 7. Changes in plasminexpression will be determined from a ratiometric plot a shown in FIG.8A. Non-parametric Anova will be used to determine statisticalsignificance.

Materials. De-identified samples from trauma patients (>25 current) andhealthy control samples (>20); clinical data; ECIS cultureware,Argatroban (thrombin inhibitor) and tranexamic acid (plasmin inhibitor)from Santa Cruz Biotech. Amicon centrifugal filter units for samplefractionation will be purchased from Millipore.

Because many gram-positive and gram-negative bacterial species canincrease the concentration of plasmin^(1A, 13A, 22A) in septic patients,we expect to experimentally determine the onset of sepsis with ECIS by aratiometric plot in the ECIS endpoints of plasma samples treated withargatroban only and those treated with argatroban and tranexamic acid(FIG. 8C). However because not all bacteria are capable of inducingfibrinolysis we expect some negative results. These types of infectionwill be used as a stratification factor in our proteomic analysisdescribed in Aim 2 c. Other confounding factors^(55A) are discussed inAim 1C. Severe trauma injuries and subsequent medical intervention mightobscure our ability to accurately measure subsequent changes to thehemostatic balance in such patients at the onset of bacterial infection.

Profile of Plasminogen Activation in Serial Samples from Septic PatientsCorrelates to ECIS Profiles.

Experimental Approach. It has been shown that plasminogen concentrationsin septic patients are significantly decreased due to bacterialinteractions that activate tissue plasminogen activator (tPA) orurokinase plasminogen activator (uPA).^(21, 51) We will screen traumapatient and healthy controls for changes in active tPA, uPA and antiplasmin in serial samples of septic and aseptic patients, to testwhether acute changes in the expression of these factors is associatedwith onset of sepsis.Materials: We will purchase: Human plasminogen total antigen assay ELISAkit (HPLGKT-TOT), Active human tPA functional assay ELISA kit (HTPAKT),Active human uPA functional assay ELISA kit (HUPAKT) Human antiplasmintotal antigen assay ELISA kit (HA2APKT-TOT), Human Thrombin AntithrombinComplex total antigen assay ELISA kit (HTATKT-COM), Active human PAI-1functional assay ELISA kit (HPAIKT) from Molecular Innovations (Novi,Mich.).

We have previously used these kits to measure active and total levels ofplasminogen activator inhibitor-1 (PA-1) in hantavirus patientsamples.^(35A) These kits are capable of detecting samples at 5 ordersof magnitude below the normal concentration ranges found in healthyhuman samples. Thus we expect to accurately quantify the concentrationsof our target analytes using appropriately diluted samples. The assayfor total human plasminogen measures plasminogen, and its proteolyticproducts plasmin and plasmin antiplasmin complex. Thus this assay willbe used to determine the concentration of plasminogen in each patient,in order to normalize the sensitivity of the ECIS measurements toputative plasmin activity in different patient samples. The assays thatmeasure active tPA and uPA are based on formation of covalent complexesbetween active tPA or uPA and PAI-1 which is immobilized on the plate.Inactive or complexed proteases will not bind to the PAI-1 and will notbe detected by the assay. This might confound data from patientsexpressing high levels of PAI-1. However such samples are expected topresent very limited plasmin activity because of PAI-1.^(52-54A) If thiswhere the case, this would likely be apparent in the clinical data, andconfirmed by PAI-1 measurement with ELISA as well as massspectrometry.^(35A)

GTPase Activation Correlates to Onset of Sepsis.

Experimental Approach. Bacteria overcome the host's defences, byhijacking RhoGTPases that regulate the actin cytoskeleton.²⁰ Thebacteria produce various toxins and virulence factors that can activateor inactivate RhoGTPases by different mechanisms, that include: a)post-translational modification of the GTPases, b) by mimicking guaninenucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) orguanine nucleotide dissociation inhibitor (GDIs) regulatory factors, c)modification of upstream regulators of small RhoGTPase.^(20A)

We have developed a rapid multiplex assay for detecting active GTPasetargets in cells exposed to patient plasma (FIG. 8).^(36A) We aretherefore uniquely positioned to screen multiple samples for evidence ofGTPase activity that can be correlated to infection. The Gtrap assay canmeasure several activated GTPases in lysates, that attach to theircognate effector proteins immobilized on up to 12 red color andfluorescence intensity encoded beads (FIG. 9A). When two different sizedbeads are used, the assay can measure up to 24-targeted conditionssimultaneously. The GST-effector chimeras consisting of the minimalGTPase binding domains (RBD) for the studies are: PAK-1 RBD, (a Rac1 andCdc42 effector), Raf-1 RBD, (a Ras effector) Rhotekin-RBD, (a Rhoeffector) RalGDS-RBD, (a RAP effector protein).^(36A)10,000 beads foreach target effector are mixed and added to cell lysates, typicallygenerated from small volume assays of 50,000 cells. The beads areincubated in cell lysates for an hr at 4° C. centrifuged and resuspendedin 50 μl buffer (1:20 final antibody dilution).

Monoclonal antibodies for each target GTPase are pooled and added to themultiplex bead suspension and incubated for an hour at 4° C. A secondaryantibody tagged with Alexa®488 dye, is then used to fluorescently tagbead-associated antibodies. The samples are then analyzed on a flowcytometer where the red fluorescence on the effector bead is used togate the green fluorescence associated with the target (FIG. 9). Atypical assay regardless of the number of GTPase targets takes only 3hrs. Therefore we are capable of analyzing many sample that are requiredfor this project.

Pathogenic bacteria have evolved to allow the adaptive development ofvirulence factors that are optimal during different stages of infection.For example it is believed that during initiation of infection,bacterial adhesins favor tissue colonization, whereas at later stages,exotoxins promote bacterial spread and blockage of immune cellresponses.^(55A) RhoGTPases are universal regulators of eukaryotic cellfunction and have been identified as primary targets of virulencefactors from diverse bacterial species^(14A, 20A) Rho GTP bindingproteins regulate the actin cytoskeleton, but they also control cellcycle progression, transcriptional activity, intracellular vesicletransport and cell transformation.^(56A) By downregulating RhoGTPases,bacterial pathogens can block crucial immune cell functions such aschemotaxis, phagocytosis and antigen presentation.^(14, 20) In ourpreliminary studies, we have sampled the GTPase activity induced bysoluble factors in the plasma of 3 septic patients (FIGS. 8 and 10). Thepatients appear to present sepsis at different stages of admission, withP14 (Staph. pneumonia) presenting as preexisting, P13 (Staph. aureus(MRSA)) within 2 days of admission and p18 a week after admission.Significantly the upregulation of RhoGTPAses in each case appears to becorrelated to the onset of sepsis. A potential pitfall in this approachis the ability to measure/infer GTPases deactivation by bacterialfactors relative to normal quiescence. The down regulation of GTPaseactivity can be measured indirectly. For P18 who suffered severeinjuries, it was surprising that RhoA activity was lower than normalcontrols (compared to P13). To test if soluble bacterial components inP18 plasma, inhibited RhoA activation (most likely by GTP hydrolysisinitiated by activation of GAPs^(57A)),^(14A) day 10 samples from P18were mixed with RhoA-activating P13 samples (examined in FIG. 8). P19(FIG. 8) samples and argatroban were used as negative, and positivecontrols respectively. The results showed that mixing P18 sample withP13 sample inhibited RhoA activation.

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Example 3

Rapid Parallel Flow Cytometry Assays of Active GTPases Using EffectorBeads.

Here we describe a rapid, bead-based effector-binding assay that canmonitor the activation status of multiple GTPases from a single celllysate derived from <100,000 cells. As proof-of-principle, wedemonstrate the utility of the assay in measuring the previouslyuncharacterized cascade of GTPases that is activated to allow cellularentry of Sin Nombre hantavirus (SNV) in the course of a productiveinfection.

Hantaviruses, which cause hemorrhagic fever with renal syndrome (HFRS)and hantavirus cardiopulmonary syndrome (HCPS), are of particularinterest because their mechanisms of host cell entry and subsequentpathogenesis are poorly understood [9B]. Viruses attach to cell surfacereceptors, promote the cytoskeletal remodeling required for breachinginter- and intracellular cellular barriers, and enter the cell throughendocytic membrane trafficking pathways; all processes thatfundamentally depend on the activation of various GTPase cascades [10B].Parallel measurements of active GTPases downstream of receptorengagement are therefore important for understanding how these signalsare regulated and how their consolidation is related to bothphysiological and pathological contexts.

Materials and Methods

Antibodies

Monoclonal rabbit anti-RAP1 (5G7): sc-47695 and rat monoclonal antiH-Ras (259): sc-35 were obtained from Santa Cruz Biotechnology, Inc(Santa Cruz, Calif.)

Monoclonal mouse antibodies: anti-Rho (A, B. C) clone 55 (#05-7788),anti-Rac1, clone 23A8, #05-389, the secondary antibody goat anti-mouseIgG (H+L) conjugated to Alexa Fluor 488, Rabbit Polyclonal anti Cdc42#07-1466 were obtained from Millipore (Temecula, Calif.). MouseMonoclonal Anti Rac1 #ARC03 was also purchased from Cytoskelton Inc.(Denver, Colo.). Mouse monoclonal anti-Rab 7 antibody was fromSigma-Aldrich (St. Louis, Mo.). Mouse monoclonal AP5 β₃ anti LIBSantibody was purchased from the Wisconsin Blood Center.

Activators and Inhibitors

Rap1 Activator 8-Cpt-2me-cAMP was used at 50 μM and from R & D Systems(Minneapolis, Minn.). Rac1 Inhibitor, NSC23766 was used at 100 μM andfrom Calbiochem Inc. (now www.emdmillipore.com). Calpeptin (Rhoactivator) was used at 0.3 μM and from Calbiochem Inc. The humanrecombinant form of the catalytic domain of p50RhoGAP from CytskeletonInc. (Denver, Colo.) was used at 2.50 μg/μl. Recombinant EGF was used at10 nM and was from Life Technologies (Carlsbad, Calif.). The novel Cdc42inhibitor CID2950007 was used at 10 μM synthesized and characterized aspreviously described [6B] and can be purchased commercially as ML141.

Buffers

RIPA buffer: 74 mM CaCl₂), 50 mM Tris-HCl pH 7.4, 1% NP-40 (v/v), 0.5%(w/v) deoxycholic acid, 0.1% (w/v) SDS, 1 mM sodium orthovanadate, andprotease inhibitors. HHB buffer: 7.98 g/L HEPES (Na salt), 6.43 g/LNaCl, 0.75 g/L KCl, 0.095 g/L MgCl₂ and 1.802 g/L glucose.

Cell Culture

HeLa and Vero E6 cells from ATCC were plated at 2×10¹ cells per well ina 48-well plate, then allowed to grow for ˜48 h to medium/low confluencein Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS,pen/strep, and L-glutamine in a 37° C. incubator with 5% CO₂.

Production of Sin Nombre Virus

SNV was propagated and titered in Vero E6 cells under strict standardoperating procedures using biosafety level 3 (BSL3) facilities andpractices (CDC registration number C20041018-0267) as previouslydescribed [11]. For preparation of UV-inactivated SNV, we placed 100 μLof virus stock (typically 1.5-2×10⁶ focus forming units/ml) in each wellof a 96-well plate and subjected the virus to UV irradiation at 254 nmfor various time intervals (˜5 mW/cm²) as described elsewhere [12B]. Weverified efficiency of virus inactivation by focus assay before removingfrom the BSL-3 facility.

Fluorescent Labeling of SNV

The envelope membrane of hantavirus particles was stained with thelipophilic lipid probe octadecylrhodamine (R18) and purified aspreviously described [12B]. The typical yield of viral preparation was1±0.5×10¹ particles/μl in 300 μl tagged with ˜10,000 R18 probes/particleor 2.7 mole % R18 probes in the envelope membrane of each particle of192 nm diameter average size [12B]. Samples were aliquoted and stored in0.1% HSA HHB buffer, and used within two days of preparation and storageat 4° C. For long-term storage, small aliquots suitable for single usewere stored at −80° C.

Effector Proteins

The GST-effector chimeras consisting of the minimal GTPase bindingdomains (RBD) used for the studies were as follows. PAK-1 RBD, a Rac1effector and Raf-1 RBD, a RAS effector protein were obtained fromMillipore. Rhotekin-RBD, a Rho effector protein was purchased fromCytoskeleton Inc. RalGDS-RBD, a RAP1 effector protein was expressed andpurified from a plasmid kindly provided by Dr. Burridge (UNC ChapelHill) [13B]. GST-RILP RBD was purified as described below.

Expression and Purification of GST-RILP

A plasmid encoding GST-RILP RBD was prepared by Daniel Cimino aspreviously described [14B]. Protein was expressed in competent E. coliBL21 cells. Cultures were grown at 37° C. to an absorbance of 0.5 O.D.measured at 595 nm and protein expression induced by transfer to roomtemperature and addition of 0.2 mMisopropyl-beta-D-1-thiogalactopyranoside (IPTG) for 16-18 h to maximizethe yield of properly folded active fusion protein. Purification ofGST-RILP was performed according to standard procedures and aspreviously described [15B] [16B]. Briefly, the harvested bacterial cellpellet was resuspended in cold PBS. Cells were lysed using lysozyme anda microtip sonicator (Misonix Inc., Newtown, Conn., U.S.A.). The celllysates were mixed with 10% Triton X-100 and mixed end over end for 30min at 4° C. Lysates were then centrifuged at 8,000×g for 10 min topellet the cellular debris. The supernatant was mixed with freshlyprepared Glutathione Sepharose 4B slurry and bound at 4° C. for 2 husing gentle agitation to keep the resin suspended in the lysatesolution. Glutathione beads with bound protein were settled using lowspeed centrifugation (500×g) followed by multiple washes before elutingthe bound GST-tagged protein with 10 mM glutathione in 50 mM Tris-HClelution buffer. Eluted protein was concentrated by passing throughMillipore Amplicon Ultra Tubes (MWCO 30,000). The purified protein wasquantified using the BCA protein estimation kit (Pierce, City, State).Single use aliquots stored at −80° C. were used in the experiments.

Preparation of GSH Beads

13 μm Superdex peptide beads were derivatized with glutathione(γ-glu-cys-gly or GSH) as previously described [7; 15; 16]. 5 μmCyto-Plex™ carboxylated beads (FM5CR0L L=1, 2, 3, . . . 12) dyed withgraded levels (L) of red emission were purchased from Thermo Scientific(City, State). The beads were then functionalized with glutathione aspreviously described [17B]. Briefly, the carboxyl functionalized beadswere converted to amine reactive N-hydroxysulfosuccinimide (Sulfo-NHS)esters using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC or EDAC) chemistry following protocols provided by the manufacturer(Thermo Scientific). The amino derivatized beads were then reacted witha bifunctional chemical crosslinker succinimidyl4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC) in order toenable subsequent attachment of GSH. 5×10 beads were suspended in 400 μlof 50 mM sodium phosphate buffer (pH 7.5) containing 0.01% Tween-20(w/v) and mixed with 8 μl of 100 mM SMCC in dimethyl sulfoxide (DMSO)and incubated with mild agitation for 30 min. The beads were centrifugedand resuspended in 360 μL of fresh buffer together with 40 μl of 200 mMGSH (pH 7) and 4 μl of 100 mM EDTA (pH 7-8). Nitrogen was bubbled slowlythrough the suspension for 5 min, the tube was capped to exclude oxygen,and the beads were gently mixed for 30 min. The beads were washed fourtimes and stored in single use aliquots in 30 mM HEPES (pH 7.5), 100 mMKCl, 20 mM NaCl, 1 mM EDTA, and 0.02% NaN₃ at 4° C.

Assembly of GST Effector Proteins on GSH Beads and GST Pull-Down Assay

Site occupancy (θ) of GSH sites on beads (limiting reagent) is regulatedby the dissociation constant K_(d) and concentration of freeglutathione-S-transferase (GST) fused to effector proteins, according toEquation (1):θ=[|GST]_(free) /K _(d))/(1+([GST]_(free) /K _(d)))  (1)

We relied on previous studies wherein the characteristic kinetic andequilibrium binding constants (K_(d)˜80 nM) of GST fused to Greenfluorescent protein (GFP) were documented [7B; 15B; 16B; 17B; 19B], toestablish optimal stoichiometric mixtures of GSH beads and specific GSTeffector fusion proteins for obtaining saturating site occupancies ofthe GST effector proteins for the present work. Typical site occupanciesof the beads at saturation are in the 1-4×10⁶ ligand sites/bead range.For example, 10,000 beads present an upper limit of 4×10¹⁰ sites or 3.3nM in 20 μL. Incubating 800 nM (10×K_(d)) of GST effector protein withthe GSH beads is expected to yield a bead site occupancy, θ, of 0.91 (or91%). In this way, known quantities of beads were mixed with effectorproteins of known concentration at the desired stoichiometry range,incubated with shaking for 2 h at 4° C., centrifuged, and resuspended inRIPA buffer at 10,000 beads/tube. Effector-bearing beads were preparedfresh for each experiment and kept on ice while cell lysates wereprepared.

Configurations of the GTPase Effector Trap Flow Cytometry Assay (G-Trap)

Vero E6 or HeLa cells were plated in 48 well plates or T 75 flasks andincubated overnight in culture media. Cells were starved for overnightin serum-free medium followed by stimulation with activator or inhibitorat concentrations and times shown in the Results section. Afterincubating cells with various inhibitors, activators, and/or SNV^(R18)for desired times, cells were lysed with 100 μL ice-cold RIPA buffer.For the purposes of standardization, a fraction of the supernatant wascollected to measure protein concentrations. Lysates were kept ice coldat all times to limit hydrolysis of active GTPases. Lysates weresonicated briefly, and then centrifuged at 14,000 rpm for 10 min toclear the lysate of any unlysed materials and DNA. For each effectorassay, 10,000 beads were added to 50 μg of protein in 100 μl of celllysate in RIPA buffer. The beads and lysate were allowed to incubate for1 h at 4° C. After incubation, the beads were pelleted in a coldcentrifuge. The bead pellets were resuspended in RIPA buffer andincubated with a primary monoclonal antibody for the target GTPase, withgentle shaking for 1 h. Depending on availability, the residual lysateswere collected and re-assayed for a different GTPase by adding theappropriate effector beads. Lysates may be saved for future assays byflash freezing in liquid nitrogen, then storing at −20° C. for up to oneweek. To minimize reporter antibody crosstalk, samples were split intoseparate fractions after lysate binding to the multiplex bead sets andthen individually probed with a reporter antibody specific for a singletarget GTPase per fraction. The beads were than centrifuged andresuspended in 10% BSA HHB buffer with an Alexa 488 secondary antibodyat a 1:20 dilution for 1 h with shaking at 4° C. Finally the beads werecentrifuged once more and resuspended in 100 μL RIPA buffer for readingon the flow cytometer.

PI-PLC Treatment

To determine whether cytoskeletal remodeling was caused by the specificinteraction of SNV^(R18) with DAF, Vero E6 cells were treated withphosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme knownto cleave GPI-anchored proteins from cell surfaces [20B; 21B].Monolayers of Vero E6 cells plated in 8-well Lab-Tek chambers werewashed with HHB and treated with 1.0 unit of PI-PLC from Bacillus cereus(Life Technologies, Carlsbad, Calif.). After incubation at 37° C. for 30min, cells were washed and resuspended in HHB buffer and analyzed at themicroscope.

GLISA

Colorimetric based, commercial kits (Cytoskeleton Inc., Denver, Colo.)for measuring Rho GTPase activation were used to measure SNV inducedactivation of Rac1 and RhoA in Vero E6 cells. About 8×10⁴ Vero E6 cellswere plated in a 12-well plate in RPMI 1640 medium. After reaching50-60% confluency, the media was replaced with starvation mediacontaining 0.5% BSA. Cells were then serum starved for 24 h beforetreatment with ˜10⁹ SNV particles at different time points. Mediaconditions included calcium (Ca²⁺), and PI-PLC (as described above).Cells were washed with ice cold PBS and then lysed and prepared forprotein assays. Positive controls included purified Rac1-GTP andRhoA-GTP provided in the kit and were used to quantify active GTPaselevels in the cell lysates. Negative controls included buffer-onlycontrols, and cell lysates prepared from quiescent control cells afterserum starvation. Cell lysates were frozen, and active Rac1 levels werequantified based on a p21-activated protein kinase binding assay [22],whereas active RhoA was quantified based on a Rhotekin binding assay[23]. All assays were performed in 96-well microtiter plates.

Stress Fiber Assay for RhoA Activation

RhoA activation was independently confirmed by monitoring actin stressfiber formation following calpeptin or SNV stimulation (1 μg/ml) for 30min. Actin filaments in control and stimulated cells were detectedfollowing paraformaldehyde (4%) fixation and Triton X-100 (0.5%)permeabilization using Alexa Fluor® 488 phalloidin (1 unit, or 0.17 μM)(Life Technologies).

Confocal Microscopy of SNV^(R18)

For live cell imaging of virus binding and endocytosis, Vero E6 cellswere plated in 8-well Lab-Tek chambers (Nunc) and temperature wasmaintained at 30° C. with an objective heater (Bioscience Tools) inappropriate buffer containing the desired cations, Ca²⁺ or Mn²⁺, whichrespectively confer low and high affinity states of α_(v)β₃[24B]. Insitu addition of SNV^(R18) was performed by micropipeting an aliquot(100 μL) of ≈1×10⁹ virus particles per well containing ≈1.5×10⁵ cells in200 μl in HBSS and mixing well by trituration with the pipet. Confocallaser scanning microscopy was performed with Zeiss META or LSM 510systems using 63×1.4 oil immersion objectives as previously described[24B]. Images were collected using 2× line averaging at 3-10 sintervals, depending on imaging time, where longer intervals werenecessary to minimize photobleaching.

Results and Discussion

Configuration and General, Applicability of G-trap Assay

Measurements of the activity of several GTPases in receptor-stimulatedcells makes use of micron-sized glutathione beads that are individuallyfunctionalized with GST-tagged cognate effector protein and specific forindividual target small GTPases. To simplify bacterial expression andpurification of effector proteins, only the GTPase binding domains (RBD)are used. Effector coated beads are incubated with cell lysates thatcontain active, GTP-bound Ras, Rho and Rab GTPases. The GTPases areselectively recruited to beads that bear the cognate effector and aredetected directly using fluorophore conjugated monoclonal antibodiesspecific for each GTPase or indirectly using secondary antibodies withfluorophore tags. It is important to note that the optimal signal-ratiobetween site occupancy of effector beads exposed to resting andactivated cell lysates is achieved by not using large excesses of celllysate proteins. This is because the effector beads are nominally usedas limiting reagents, and therefore can approach saturation even in theresting cell lysate. This would obscure the accurate detection ofincreased activity status of GTPases in stimulated cell lysates.Impirically, we found that 10,000 beads and 50 μg protein in 100 μl wereoptimal for each effector assay (see methods).

In multiplex configuration distinct effectors were immobilized onCytoplex™ beads of graded fluorescence intensities of a fluorophore witha fixed red wavelength of 700 nm [19B](FIG. 11). An extra set ofeffector free beads was used as a control for nonspecific binding (e.g.L4 in FIG. 11B. To assess the degree of nonspecific reporter antibodycrosstalk during the labeling step, we measured the levels of binding tounconjugated GSH beads using a mixture of reporter antibodies for RhoA,Rac, Cdc42, H-Ras and Rap1 GTPases under the following three conditions:a) GSH beads mixed with all antibodies and measurement of the aggregatefluorescence of nonspecifically bound antibodies (multiplex in FIG. 1D),b) individual bead populations were treated with each antibodyseparately and measured separately (fraction in FIG. 11D), c) individualbead populations were treated with each antibody separately, thencombined and measured as a single mixture (mixed fraction in FIG. 11D).For the five GTPase antibodies tested, the multiplex sample wascomparable to fraction and mixedfraction samples for the Rac1, RhoA, andRap1 antibodies but higher for Cdc42 and H-Ras antibodies. The degree ofnon-specific binding depends on antibody type, batch and supplier.

Therefore, it is important for the user to be familiar with bindingcharacteristics of new antibody batches before using them in multiplexformat. In the present application, non-specific binding values shown inFIG. 11D, were significantly lower (≥5 fold to ≥30 fold) thanfluorescence signals associated with resting and activated cells,depending on assay conditions (cf. FIG. 12). In these circumstanceserrors from non-specific binding are trivial. However, in cases were thefluorescence signals associated with specific GTPase-activity assays areof comparable order of magnitude to nonspecific background readings(e.g. NSC23766 or ML141 treated cells in FIG. 12), it is important tomodify the multiplex protocol. This can be addressed by adopting themixed fraction format, where multiplexed samples that are recovered fromthe lysates are partitioned into fractions that correspond to the numberof target analytes, and stained separately with reporter antibodies. Inthe case of the example shown in FIG. 1B-D, Cdc42 and Rap1, may bestained with reporter antibodies as separate fractions, while Rac1, RhoAand HRas can be treated as a full multiplex. In summary it is importantfor the user to be familiar with binding characteristics of antibodybatches before using them in a format of the G-trap assay that is mostsuitable for the prevailing conditions.

G-trap Assay Validation

Cdc42, Rac1 and RhoA are among the most well characterized members ofthe Rho subfamily of GTPases. They are known for orchestratingcytoskeletal reorganization dynamics (filamentous actin and myosin 2)and crosstalk to antagonize each other's activities [25B; 26B; 27B;28B]. The availability of specific regulators of their activity makesthese GTPases a suitable platform to test the applicability of ourG-trap assay. HeLa cells were treated with the following signalingregulators: 1) NSC23766, a specific inhibitor of Rac1 interaction withits two upstream GEFs Tiam1 and Trio (Trio is also identified as a RhoAtarget [29B: 30B]), 2) CID2950007, a novel Cdc42 specific inhibitor [6B]commercially sold as ML141, and 3) calpeptin, an upstream activator ofRhoA [31B]. Cdc42, Rac1 and RhoA were activated by adding 10 nM EGF tocells. Readings were taken at 3 min and 20 min after stimulation. Thedata were corrected for nonspecific binding by subtracting thefluorescent antibody binding to the GST-conjugated control beads fromthe antibody binding to GST-effector bearing beads. EGF-stimulated cellsamples were normalized to unstimulated resting control cell samples.Data were collected 3 min (FIG. 12A, 12C) and 20 min (FIGS. 12B and 12D)post-EGF-stimulation. RhoA data were separately paired with Rac1 andCdc42 for ease of comparison of the putative antagonists. The activationof RhoA lagged behind both Rac1 and Cdc42 activation, at 3 min andsupplanted both Cdc42 and Rac1 at 20 min. At this juncture it is not yetclear whether the time-dependent dominance in the relative amplitudes ofRac1 and RhoA are due to their mutual antagonism. Cell treatment withthe Rac1-GEF inhibitor. NSC23766, suppressed Rac1 while RhoA activitywas elevated relative to resting cells. Treatment of cells with EGF andNSC23766 in combination resulted in a dramatic activation of RhoA(>5-fold relative to resting cells) that was well above the activationof RhoA observed following EGF-stimulation alone. Calpeptin-mediatedactivation of RhoA was tested for comparison and found to be accompaniedby a concomitant decrease in Rac1 activity. Collectively, these data areconsistent with the known mutual antagonism of Rac1 and RhoA [25B; 32B];wherein the inhibition of Rac1 is sensed and results in amplified RhoAactivity in the absense of a counteracting Rac1 effect. The targetedactivity of NSC23766 against Rac1 and not RhoA suggests that Trio is notan important upstream factor for RhoA activation under our experimentalconditions. Interestingly, though Rac1 and Cdc42 are believed to havepartially overlapping functions in mediating the formation of actin-richprotrusions in migrating cells [2B; 33B], the total inhibition of Cdc42had no effect on RhoA activity under our experimental conditions (FIGS.12C and 12D) as previously shown [6B]. These results suggest that underour experimental conditions, the up or downstream effector signalsassociated with Cdc42 do not impinge on or regulate RhoA and vice versa.Together the data demonstrate the power of the G-trap assay insensitively and temporally dissecting GTPase responses to stimuli andpharmacologic manipulation.

Monitoring Virus-Induced GTPase Activation: Morphologic and BiochemicalAssessment

Exposure of Vero E6 cells to known titers of UV killed and fluorescentlylabeled SNV particles causes cells to contract, lose cell contacts, andform filopodia, lamellipodia and stress fibers (FIG. 13A-F). Ultimatelythe virus is internalized and present in Rab7 positive late endosomes.Such morphological changes and membrane trafficking events areanticipated to be associated with the changes in the activities ofmultiple GTPases. Actin based structure changes are associated withCdc42 (filopodia), Rac1 (lamellipodia), and RhoA (stress fiber formationand cellular contraction) [34B]. Staining for actin with Alexa Fluor488® phalloidin, identified RhoA induced F-actin stress fibers formed inresponse of treatment of resting cells with calpeptin (compare FIG. 13Dvs. 13E) were similar to the stress fibers formed in response to SNVexposure (FIG. 13F). The ruffling observed in SNV-treated cells issimilar to that seen in response to EGF-mediated activation ofRac-dependent ruffling [35B-B]. The loss of cell adhesion is connectedto integrin affinity regulation, which is presumed to be downstream ofantagonsitic Rap and H-Ras activity [36B] [37B]. AP-5 monoclonalantibodies recognize the PSI domain of extended conformation activatedβ₃ integrins at the cell surface [38B] and enabled detection of theonset of cellular detachment at the cell junctions in virus treatedcells; based on poor AP5 staining of the plasma membrane and instead astrong perinuclear substratum staining (arrows in FIG. 13I). See forcomparison resting and Mn²⁺ stimulated (maximal cell surface integrinactivation) controls (FIG. 13G-H). All of the described morphologicindicators of changes in GTPase activity prompted us to use conventionalGLISAs in comparision with our G-trap assay for monitoring theactivation status of individual GTPases in SNV treated cells.

We first used GLISA assays to measure Rho GTPase activation as afunction of virus exposure time of wild-type Vero cells and cells wheresurface-expressed DAF was first cleaved with PI-PLC [20B] (FIG. 4).Virus treatment resulted in a robust activation of first RhoA at 3 minand subsequently Rac1 at 7 min with both falling to baseline levels by30 min. After 60 min of virus exposure the levels of active GTP-Rho and-Rac fell below baseline levels, most likely because the expression ofGTP-bound Rho GTPases is regulated by cell-cell and cell-matrix adhesion[33B] [39B]. In DAF-cleared cells, Rac failed to be activated across alltimepoints indicating that virus engagement of a GPI anchored receptorsuch as DAF was required upstream of Rac1 activation. It is important tonote that, the GLISA assays were performed separately on different daysusing different cell lysates, therefore the relative amplitudes of RhoAand Rac1 can not be accurately correlated to their temporal responses inthe same way that the G-trap data shown in FIG. 12 was analyzed.

We next turned to the G-trap assay to examine the induction Rho proteinactivity in virus treated Vero E6 cells. Using known Rho GTPaseactivators and inhibitors the assay validation experiments (FIG. 12B)and results were recapitulated using permissive Vero E6 cells. In virusstimulated cell lysates (FIG. 14A) and analogous to the GLISA results,RhoA activity increased ≥4-fold above resting levels within 3 minpost-treatment. Active Rac1 levels also increased to a lesser extent atthe 3 min timepoint. At the 20 min timepoint timepoint, the activity ofboth GTPases decrease to baseline levels confirming the trend documentedby GLISA above (compare FIG. 4 vs. FIG. 14A). The temporal overlap inthe activation of RhoA and Rac1 implies that SNV binding inducessignaling cascades that lie upstream of RhoA and Rac1 activation, whichmirrors what has been observed in a study on Group BCoxsackievirus-activated cells [40B]. Rac1 and Rho normally mutuallysuppress each other's activity in response to single stimuli as affirmedin 12 and elsewhere in the literature [2B; 25B; 41B]. Their apparentco-stimulation by the virus may reflect the activation of multiplesignaling cascades through sequential events with distinct spatial andtemporal characteristics [32]. This notion is further supported by thesimultaneous visualization of multiple GTPase biosensors during cellmigration where surprisingly RhoA was activated exclusively near thecell edge connected with leading-edge advancement. In contrast, Cdc42and Rac1 were activated away from the leading edge with a delay of ˜40seconds [25B].

The potential roles of the mutually antagonistic Rap1 and H-Rasdownstream of SNV signals that lead to loss in cell adhesion were alsoexamined by G-trap assay. Beads functionalized with effectors for Rap(GST RalGDS-RBD) and H-Ras (Raf-1 RBD) were used to simultaneously assayactivated Rap1, and H-Ras GTPases from cell lysates of virus treatedcells. An activator of Epac/Rap1, 8-Cpt-2m-cAMP, served as a specificitycontrol. Activation of Rap1 by 8-Cpt-2m-cAMP was readily demonstratedand as expected decreased basal levels of H-Ras in the G-trap assay[42B]. The activity of Rap1 and H-Ras in virus-activated cell lysates at3 min and 20 min post-exposure showed that the GTPase pair was activatedsequentially, where active Rap 1 peaked within the first 3 min andsubsequently declined at 20 min. H-Ras remained low while Rap1 was atits zenith, eventually peaking at 20 min as active Rap1 was in decline.The coincidence between high H-Ras activity and loss of cell-celladhesion might be linked to the proclivity of H-Ras to suppress integrinactivity [43B] (cf. FIG. 13F).

Finally, the activation status of GTPases involved in the intracellulartransport of virus particles following internalization, were assessed bymonitoring the levels of active Rab7, a GTPase associated with early tolate endosome and lysosomal transport [14B; 44B; 45B]. Active, GTP-boundRab7 levels peaked 15 min following virus exposure (FIG. 14B). Thetiming of maximal Rab7 coincides with the timing of fluorescentlylabeled SNV delivery to perinuclear late endosomes traced by microscopy.The time frame is also within the range reported for the delivery ofother large viral cargoes to Rab7-positive late endosomes [46B; 47B].

In sum, we have devised a novel, cost effective, flowcytometry-compatible, bead-based effector-binding assay (G-trap) forrapidly monitoring the activation status of multiple members of the RasGTPase superfamily in cell lysates. We have validated the assay throughthe use of known agonists and antagonists of individual GTPases. Thestudy provides previously unknown mechanistic detail about SNV inducedactivation of cellular signaling to promote virus entry and transport.We have recently used a novel inhibitor to define the involvement ofCdc42 GTPase in the lifecycle of Sin Nombre virus infection [6B].Selective pharmacological inhibitors of the active GTPases can be pairedwith the G-trap assay to further define the signaling pathways that areimportant for regulating the lifecycles of pathogens that rely on thesame processes. In similar vein we have shown that Y27632, the inhibitorof RhoA kinase (ROCK), blocks the loss of cell barrier function inpolarized endothelial cells and thus limits viral infection (Buranda,unpublished results). We therefore anticipate that this approach willenable investigations into the interconnection of signaling networks viaGTPase cascades in normal cellular functions as compared to viral orbacterial infections or other pathogenic processes.

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What is claimed is:
 1. A kit comprising: (a) a population of beads whichhave two or more sizes, which are labeled with a first fluorophorehaving a single wavelength and a plurality of intensity levels and whichare covalently coupled to a plurality of effector proteins which bind tocognate, infection-associated-guanosine triphosphate hydrolases(GTPases) to form effector protein-infection-associated-GTPaseconjugates, wherein said GTPases are ras-related C3 botulinum toxinsubstrate 1 (Rac1) and Ras-related protein 1 (Rap 1) and said effectorproteins which bind to Rac is 1 are selected from the group consistingof p21 activated kinase 1 rho binding domain (PAK-1 RBD), steroidreceptor RNA activator 1 (Sra1), insulin receptor tyrosine kinasesubstrate p58/53 (IRSp58/53), p21 activated kinase 1 (PAK1), p21activated kinase 2 (PAK2), p21 activated kinase 3 (PAK3) and mixturesthereof and said effector protein which binds to Rap1 is ra1 guaninenucleotide dissociation stimulator rho binding domain (Ra1GDS-RBD); (b)GTPase-specific antibodies which bind to said effectorprotein-infection-associated-GTPase conjugates formed on the beads; (c)detector antibodies which are specific to the GTPase-specific antibodiesand which are labeled with a second fluorophore having a wavelength(color) which is different from that of the first fluorophore; and (d)optionally, instructions with respect to the use of the beads,GTPase-specific antibodies and detector antibodies in a flow cytometryassay for detecting the existence or absence of sepsis in a patient orsubject in need.
 2. The kit of claim 1, wherein: (a) the effectorproteins are PAK-1 RBD and RalGDS-RBD (a; and (b) the first fluorophoreis a red fluorophore and the second fluorophore is a green fluorophore.3. The kit of claim 2, wherein said red fluorophore is a red fluorescentdye and the green fluorophore is a green fluorescent dye.
 4. The kit ofclaim 1, wherein the first fluorophore is a fluorescent dye and thesecond fluorophore is a fluorescent dye.
 5. The kit of claim 1, wherein:(a) the population of beads is defined by distinct sub-sizes, each ofwhich conducts a unique assay; and (b) the beads are internallycovalently coupled to red and infrared fluorophores with differentintensities.
 6. The kit of claim 1, wherein the beads are microbeads. 7.The kit of claim 1, wherein the kit contains at least 10,000 beadsconjugated to said effector protein.
 8. The kit of claim 1, wherein thebeads are defined by at least two distinct sub-sizes.
 9. The kit ofclaim 1, wherein the kit can be used in conjunction with a multiplexflow cytometry assay which processes 96 or 384 well-plates.
 10. The kitof claim 1, wherein the kit comprises a positive or negative control.11. The kit of claim 1, wherein said flow cytometry assay is a multiplexflow cytometry assay.
 12. A kit comprising: (a) a population of beadswhich have two or more sizes, which are labeled with a first fluorophorehaving a single wavelength and a plurality of intensity levels and whichare covalently coupled to a plurality of effector proteins which bind tocognate, infection-associated-guanosine triphosphate hydrolases(GTPases) to form effector protein-infection-associated-GTPaseconjugates, wherein said GTPases are ras-related C3 botulinum toxinsubstrate 1 (Rac1) and Ras-related protein 1 (Rap 1) and said effectorprotein which binds to Rac 1 is p21 activated kinase 1 rho bindingdomain (PAK-1 RBD) and said effector protein which binds to Rap1 is ra1guanine nucleotide dissociation stimulator rho binding domain(Ra1GDS-RBD); (b) GTPase-specific antibodies which bind to said effectorprotein-infection-associated-GTPase conjugates formed on the beads; (c)detector antibodies which are specific to the GTPase-specific antibodiesand which are labeled with a second fluorophore having a wavelength(color) which is different from that of the first fluorophore; and (d)instructions with respect to the use of the beads, GTPase-specificantibodies and detector antibodies in a multiplexed flow cytometry assayfor detecting the existence or absence of sepsis in a patient or subjectin need.